# MIX-MB(B) — Biotransformation

## Biotransformation Process Standards

This document identifies Minimum Information (MI) required to report the biotransformation process between microorganisms and xenobiotic compounds. MIX-MB(B) bridges MIX-MB(X) (xenobiotics) and MIX-MB(M) (microbes) by defining standards for the experimental assays, measurements, and outcomes.

**Author:** Mahnoor Zulfiqar **Part of:** MIX-MB Standard\_main\
**Compatible with:**

* MIX-MB(X)
* MIX-MB(M) **Alignment:** ChEMBL, Bioschemas, BioAssay Ontology (BAO), FAIR principles

***

***

## 1. Overview

MIX-MB(B) establishes minimum information standards for documenting biotransformation experiments where microorganisms metabolize xenobiotic compounds. This standard ensures:

* **Reproducibility:** Complete experimental protocols and conditions
* **Comparability:** Standardized measurements and reporting
* **Traceability:** Links between substrates, organisms, and products
* **Quality:** Validation and quality control measures

It bridges MIX-MB(X) (xenobiotics) and MIX-MB(M) (microbes):

```
MIX-MB(X)              MIX-MB(B)              MIX-MB(M)
[Xenobiotics] -----> [Biotransformation] <----- [Microbes]
   Substrate              Process               Organism
   Product                                      Enzyme
   Structure             Activity               Assay
```

### 1.4 Identifiers and Cross-Referencing

**This is the first practical step: establish the three-way identifier link (RIDX → CIDX → AIDX) before recording any activity.**

#### The Three-Way Identifier System

Every biotransformation event in MIX-MB is anchored by three identifiers that must be consistent across all submission files:

| Identifier | Entity                          | Appears in                                     | Format example                                          |
| ---------- | ------------------------------- | ---------------------------------------------- | ------------------------------------------------------- |
| **RIDX**   | Reference (study / publication) | All files                                      | `Zimmermann_GutBiotransformationAtlas`                  |
| **CIDX**   | Compound (substrate or product) | `COMPOUND_RECORD.tsv`, `ACTIVITY.tsv`          | `CIDX0001`                                              |
| **AIDX**   | Assay (organism × condition)    | `ASSAY.tsv`, `ASSAY_PARAM.tsv`, `ACTIVITY.tsv` | `Zimmermann_Actinomyces_graevenitzii_biotransformation` |

Every row in `ACTIVITY.tsv` must carry all three identifiers. They are the join keys that link compounds to assays to publications — if any one is missing or inconsistent, the record cannot be deposited or queried.

#### Reference Index (RIDX)

The RIDX is assigned once per study and used unchanged across every file in the submission.

**Minting rules:**

* Format: `[FirstAuthorLastName]_[DescriptiveLabel]`, e.g. `Zimmermann_GutBiotransformationAtlas`
* Must be unique across all submissions to the target database
* Use only ASCII letters, digits, underscores, and hyphens — no spaces or special characters
* Set the RIDX at the outset of data preparation; do not change it after any file references it

#### Activity Record Cross-Referencing

Individual rows in `ACTIVITY.tsv` do not require their own unique identifier, but every row must carry a consistent `CIDX × AIDX × RIDX` triplet. Each such triplet represents one compound–assay interaction.

**Rules:**

* The same `CIDX × AIDX` pair may appear on multiple rows if multiple activity types are reported (e.g. both `Substrate` and `Product` for the same compound in the same assay)
* Do not reuse the same `CIDX × AIDX × ACTION_TYPE` combination for separate measurements — add a distinguishing note in `ACTIVITY_COMMENT` instead
* Every CIDX referenced in `ACTIVITY.tsv` must exist in `COMPOUND_RECORD.tsv`; every AIDX must exist in `ASSAY.tsv` — orphan references will fail ChEMBL validation

#### Minting Scheme for Unconfirmed Biotransformation Events

| Situation                                           | `ACTION_TYPE` | CIDX to use           | Notes                                                     |
| --------------------------------------------------- | ------------- | --------------------- | --------------------------------------------------------- |
| Substrate consumed (confirmed)                      | `Substrate`   | Known `CIDX[nnnn]`    | Quantitative `VALUE` recommended                          |
| Product identified (MSI Level 1–2)                  | `Product`     | Known `CIDX[nnnn]`    | InChIKey required in `COMPOUND_RECORD.tsv`                |
| Product putatively characterised (MSI Level 3)      | `Product`     | `PUTATIVE_[RIDX]_[n]` | Add structural class and MSI level in `ACTIVITY_COMMENT`  |
| Product detected, unknown structure (MSI Level 4–5) | `Product`     | `UNKNOWN_[RIDX]_[n]`  | Add m/z, adduct type, and MSI level in `ACTIVITY_COMMENT` |
| No biotransformation detected                       | `No Activity` | Substrate CIDX        | Set `TEXT_VALUE` to `"No biotransformation detected"`     |

For time-course or dose-response experiments, group multiple measurements under the same `CIDX × AIDX` pair and distinguish individual rows using `ACTIVITY_COMMENT` (e.g. timepoint or concentration) rather than minting new identifiers.

***

## 2. Bioschemas

MIX-MB(B) uses Bioschemas profiles to provide structured, FAIR-compliant annotation of the experimental study, laboratory protocol, and results dataset. These profiles cross-link with the organism metadata in MIX-MB(M) (Taxon, Sample profiles) and the chemical metadata in MIX-MB(X) (MolecularEntity profile). Bioschemas fulfils the Findability and Interoperability aspects of FAIR.

The following profiles are used:

| Profile                                                              | Source     | Version     | Use in MIX-MB(B)                       |
| -------------------------------------------------------------------- | ---------- | ----------- | -------------------------------------- |
| [Study](https://bioschemas.org/profiles/Study/0.3-DRAFT)             | Bioschemas | 0.3-DRAFT   | Overall experiment design and metadata |
| [LabProtocol](https://bioschemas.org/profiles/LabProtocol/0.7-DRAFT) | Bioschemas | 0.7-DRAFT   | Biotransformation assay protocol       |
| [Dataset](https://bioschemas.org/profiles/Dataset/1.0-RELEASE)       | Bioschemas | 1.0-RELEASE | Activity and measurement results       |

***NOTE:*** Study and LabProtocol profiles map to `ASSAY.tsv` and `ASSAY_PARAM.tsv` for ChEMBL submission. Dataset covers activity data in `ACTIVITY.tsv` and supports deposition in MetaboLights or GNPS.

***

### 2.1 Study Profile

Use [Bioschemas Study 0.3-DRAFT](https://bioschemas.org/profiles/Study/0.3-DRAFT) to describe the overall biotransformation experiment, linking the xenobiotic substrate, microbial organism, and assay conditions into a single structured record.

**Minimum Properties:**

| Property         | Expected Type            | Cardinality | Description                                                |
| ---------------- | ------------------------ | ----------- | ---------------------------------------------------------- |
| `@context`       | URL                      | ONE         | `"https://schema.org/"`                                    |
| `@type`          | Text                     | ONE         | `"Study"`                                                  |
| `@id`            | IRI                      | ONE         | Globally unique IRI identifying this study                 |
| `dct:conformsTo` | IRI                      | ONE         | `"https://bioschemas.org/profiles/Study/0.3-DRAFT"`        |
| `identifier`     | PropertyValue, Text, URL | ONE         | Study identifier (e.g., RIDX, MetaboLights accession, DOI) |
| `name`           | Text                     | ONE         | Study title                                                |

**Recommended Properties:**

| Property        | Expected Type        | Cardinality | Description                                                          |
| --------------- | -------------------- | ----------- | -------------------------------------------------------------------- |
| `description`   | Text                 | ONE         | Free-text description of the biotransformation study                 |
| `author`        | Organization, Person | MANY        | Principal investigator(s) and contributing authors                   |
| `datePublished` | Date                 | ONE         | Publication or submission date                                       |
| `url`           | URL                  | ONE         | Link to the study in a public repository (e.g., MetaboLights)        |
| `studyDomain`   | Text                 | MANY        | Research domains: `"microbiology"`, `"xenobiotic biotransformation"` |

**Optional Properties (MIX-MB(B) relevant):**

| Property               | Expected Type          | Cardinality | Description                                                  |
| ---------------------- | ---------------------- | ----------- | ------------------------------------------------------------ |
| `citation`             | CreativeWork, URL      | MANY        | Related publications                                         |
| `keywords`             | DefinedTerm, Text      | MANY        | Descriptive keywords (e.g., gut microbiome, drug metabolism) |
| `measurementTechnique` | DefinedTerm, Text, URL | MANY        | Analytical technique (e.g., LC-MS/MS, CHMO:0000470)          |
| `variableMeasured`     | PropertyValue, Text    | MANY        | Primary outcome variable (e.g., substrate depletion %)       |
| `isPartOf`             | CreativeWork, URL      | MANY        | Larger project or dataset collection this study belongs to   |

**Example:**

```json
{
  "@context": "https://schema.org/",
  "@type": "Study",
  "@id": "https://www.ebi.ac.uk/metabolights/MTBLS1234",
  "http://purl.org/dc/terms/conformsTo": {
    "@id": "https://bioschemas.org/profiles/Study/0.3-DRAFT",
    "@type": "CreativeWork"
  },
  "identifier": "MTBLS1234",
  "name": "Microbial biotransformation of NSAIDs by human gut bacteria under anaerobic conditions",
  "description": "Whole-cell biotransformation screen of 12 NSAIDs against 25 human gut bacterial strains, measuring substrate depletion and product formation by LC-MS/MS over 24 hours",
  "author": [
    { "@type": "Person", "name": "Mahnoor Zulfiqar", "affiliation": "EMBL Heidelberg" }
  ],
  "datePublished": "2026-03-05",
  "url": "https://www.ebi.ac.uk/metabolights/MTBLS1234",
  "studyDomain": ["microbiology", "xenobiotic biotransformation"],
  "measurementTechnique": {
    "@type": "DefinedTerm",
    "name": "liquid chromatography-mass spectrometry",
    "termCode": "CHMO:0000470"
  },
  "variableMeasured": "substrate depletion percentage",
  "keywords": ["gut microbiome", "drug biotransformation", "NSAID metabolism", "LC-MS/MS"]
}
```

***NOTE:*** `identifier` from this profile maps to the `RIDX` field across all ChEMBL submission files (`REFERENCE.tsv`, `ASSAY.tsv`, `ACTIVITY.tsv`).

***

### 2.2 LabProtocol Profile

Use [Bioschemas LabProtocol 0.7-DRAFT](https://bioschemas.org/profiles/LabProtocol/0.7-DRAFT) to document the biotransformation assay protocol — covering inoculation, substrate exposure, incubation conditions, sampling, and analytical extraction.

**Minimum Properties:**

| Property         | Expected Type | Cardinality | Description                                                                      |
| ---------------- | ------------- | ----------- | -------------------------------------------------------------------------------- |
| `@context`       | URL           | ONE         | `"https://schema.org/"`                                                          |
| `@type`          | Text          | ONE         | `"LabProtocol"`                                                                  |
| `@id`            | IRI           | ONE         | Globally unique IRI (e.g., protocols.io DOI)                                     |
| `dct:conformsTo` | IRI           | ONE         | `"https://bioschemas.org/profiles/LabProtocol/0.7-DRAFT"`                        |
| `purpose`        | Text          | ONE         | Purpose of the protocol (e.g., `"Anaerobic whole-cell biotransformation assay"`) |
| `url`            | URL           | ONE         | Link to the protocol (protocols.io, supplementary material, etc.)                |

**Recommended Properties:**

| Property        | Expected Type                                     | Cardinality | Description                                            |
| --------------- | ------------------------------------------------- | ----------- | ------------------------------------------------------ |
| `name`          | Text                                              | ONE         | Protocol name                                          |
| `identifier`    | PropertyValue, Text, URL                          | ONE         | Protocol identifier (e.g., protocols.io accession)     |
| `description`   | Text                                              | ONE         | Short description of the method                        |
| `instrument`    | Text, URL                                         | MANY        | Equipment used (e.g., anaerobic chamber, LC-MS system) |
| `reagent`       | BioChemEntity, ChemicalSubstance, MolecularEntity | MANY        | Substrates, solvents, and reagents used                |
| `bioSampleUsed` | BioSample, Sample, URL                            | MANY        | Microbial cultures (links to MIX-MB(M) Sample records) |
| `labEquipment`  | DefinedTerm, Text, URL                            | MANY        | Specific equipment                                     |

**Optional Properties (MIX-MB(B) relevant):**

| Property            | Expected Type                | Cardinality | Description                                  |
| ------------------- | ---------------------------- | ----------- | -------------------------------------------- |
| `duration`          | Text, URL                    | ONE         | Total protocol duration (e.g., `"24 hours"`) |
| `step`              | HowToSection, HowToStep, URL | MANY        | Sequential protocol steps                    |
| `computationalTool` | SoftwareApplication          | MANY        | Software used in the analysis pipeline       |
| `citation`          | CreativeWork, URL            | MANY        | Literature supporting this protocol          |
| `isPartOf`          | LabProtocol, URL             | MANY        | Parent protocol this is a sub-protocol of    |

**Example:**

```json
{
  "@context": "https://schema.org/",
  "@type": "LabProtocol",
  "@id": "https://dx.doi.org/10.17504/protocols.io.xyz123",
  "http://purl.org/dc/terms/conformsTo": {
    "@id": "https://bioschemas.org/profiles/LabProtocol/0.7-DRAFT",
    "@type": "CreativeWork"
  },
  "name": "Anaerobic whole-cell biotransformation assay with gut bacteria",
  "purpose": "Quantify xenobiotic biotransformation by gut bacterial strains under physiological anaerobic conditions",
  "identifier": "protocols.io:xyz123",
  "url": "https://dx.doi.org/10.17504/protocols.io.xyz123",
  "description": "Stationary-phase cultures are washed, resuspended at OD₆₀₀ 1.0, and incubated with xenobiotic at 37°C under strict anaerobic conditions for 24 hours. Substrate depletion and product formation are quantified by LC-MS/MS.",
  "instrument": [
    "Anaerobic chamber (Coy Laboratory Products)",
    "Agilent 1290 UHPLC",
    "Thermo Q Exactive Plus Orbitrap MS"
  ],
  "reagent": [
    { "@type": "ChemicalSubstance", "name": "Ibuprofen", "identifier": "CHEMBL1201246" },
    { "@type": "ChemicalSubstance", "name": "DMSO (vehicle)", "identifier": "CHEBI:28262" },
    { "@type": "ChemicalSubstance", "name": "BHI medium supplemented with hemin and vitamin K1" }
  ],
  "bioSampleUsed": {
    "@type": "Sample",
    "identifier": "biosample:SAMN02604091",
    "name": "Escherichia coli K-12 MG1655 anaerobic culture"
  },
  "duration": "24 hours",
  "step": [
    { "@type": "HowToStep", "name": "Culture preparation", "text": "Grow bacteria to stationary phase in BHI medium under anaerobic conditions" },
    { "@type": "HowToStep", "name": "Cell harvesting", "text": "Centrifuge at 5000 × g, 10 min, 4°C; wash twice with sterile PBS" },
    { "@type": "HowToStep", "name": "Substrate addition", "text": "Add xenobiotic to final concentration of 100 µM (0.1% DMSO)" },
    { "@type": "HowToStep", "name": "Incubation", "text": "Incubate at 37°C in anaerobic chamber for 24 h with sampling at 0, 3, 6, 12, 24 h" },
    { "@type": "HowToStep", "name": "Sample quenching", "text": "Add equal volume ice-cold acetonitrile with 0.1% formic acid; centrifuge at 15,000 × g" },
    { "@type": "HowToStep", "name": "LC-MS/MS analysis", "text": "Analyse supernatant by reversed-phase LC-MS/MS in negative ion mode" }
  ]
}
```

***NOTE:*** This profile maps to `ASSAY.tsv` (`ASSAY_DESCRIPTION`, `ASSAY_TYPE`) and `ASSAY_PARAM.tsv` (incubation conditions, atmosphere, duration) for ChEMBL submission.

***

### 2.3 Dataset Profile

Use [Bioschemas Dataset 1.0-RELEASE](https://bioschemas.org/profiles/Dataset/1.0-RELEASE) to describe the biotransformation activity data — making results findable and citable as a distinct, structured dataset linked to the study and compounds.

**Minimum Properties:**

| Property         | Expected Type            | Cardinality | Description                                             |
| ---------------- | ------------------------ | ----------- | ------------------------------------------------------- |
| `@context`       | URL                      | ONE         | `"https://schema.org/"`                                 |
| `@type`          | Text                     | ONE         | `"Dataset"`                                             |
| `@id`            | IRI                      | ONE         | Globally unique IRI (e.g., MetaboLights URL, DOI)       |
| `dct:conformsTo` | IRI                      | ONE         | `"https://bioschemas.org/profiles/Dataset/1.0-RELEASE"` |
| `name`           | Text                     | ONE         | Dataset name                                            |
| `description`    | Text                     | ONE         | Description of the activity dataset contents            |
| `identifier`     | PropertyValue, Text, URL | ONE         | Dataset identifier (e.g., MetaboLights accession, DOI)  |
| `keywords`       | DefinedTerm, Text        | MANY        | Keywords for discovery                                  |
| `license`        | CreativeWork, URL        | ONE         | Dataset license (e.g., CC BY 4.0)                       |
| `url`            | URL                      | ONE         | URL where the dataset can be accessed                   |

**Recommended Properties:**

| Property               | Expected Type          | Cardinality | Description                                                           |
| ---------------------- | ---------------------- | ----------- | --------------------------------------------------------------------- |
| `creator`              | Organization, Person   | MANY        | Dataset authors                                                       |
| `citation`             | CreativeWork, URL      | MANY        | Publication associated with the dataset                               |
| `measurementTechnique` | DefinedTerm, Text, URL | MANY        | Analytical method (e.g., LC-MS/MS)                                    |
| `variableMeasured`     | PropertyValue, Text    | MANY        | Variables in the dataset (substrate depletion, product concentration) |
| `version`              | Text                   | ONE         | Dataset version                                                       |
| `distribution`         | DataDownload           | MANY        | Download links for the data files                                     |
| `encodingFormat`       | Text, URL              | MANY        | File format(s) (e.g., `"text/tab-separated-values"`)                  |

**Optional Properties (MIX-MB(B) relevant):**

| Property           | Expected Type     | Cardinality | Description                                               |
| ------------------ | ----------------- | ----------- | --------------------------------------------------------- |
| `isBasedOn`        | CreativeWork, URL | MANY        | Raw data files or preceding datasets this is derived from |
| `isPartOf`         | CreativeWork, URL | MANY        | Larger study or repository collection                     |
| `hasPart`          | CreativeWork, URL | MANY        | Component datasets (e.g., raw mzML, processed TSVs)       |
| `temporalCoverage` | Text              | ONE         | Date range of experiments                                 |
| `sameAs`           | URL               | MANY        | Same dataset in another repository                        |

**Example:**

```json
{
  "@context": "https://schema.org/",
  "@type": "Dataset",
  "@id": "https://www.ebi.ac.uk/metabolights/MTBLS1234",
  "http://purl.org/dc/terms/conformsTo": {
    "@id": "https://bioschemas.org/profiles/Dataset/1.0-RELEASE",
    "@type": "CreativeWork"
  },
  "name": "Biotransformation activity data — NSAIDs × gut bacteria screen",
  "description": "ChEMBL-formatted activity data (ACTIVITY.tsv, ASSAY.tsv) for the biotransformation of 12 NSAIDs by 25 human gut bacterial strains, quantified by LC-MS/MS substrate depletion and product formation.",
  "identifier": "MTBLS1234",
  "keywords": ["biotransformation", "gut microbiome", "NSAID", "LC-MS/MS", "substrate depletion"],
  "license": "https://creativecommons.org/licenses/by/4.0/",
  "url": "https://www.ebi.ac.uk/metabolights/MTBLS1234",
  "creator": [
    { "@type": "Person", "name": "Mahnoor Zulfiqar", "affiliation": "EMBL Heidelberg" }
  ],
  "citation": {
    "@type": "CreativeWork",
    "identifier": "https://doi.org/10.xxxx/example",
    "name": "Systematic screen of gut microbial drug biotransformation"
  },
  "measurementTechnique": {
    "@type": "DefinedTerm",
    "name": "liquid chromatography-mass spectrometry",
    "termCode": "CHMO:0000470"
  },
  "variableMeasured": [
    { "@type": "PropertyValue", "name": "substrate_depletion", "unitText": "%" },
    { "@type": "PropertyValue", "name": "product_concentration", "unitText": "µM" }
  ],
  "distribution": [
    {
      "@type": "DataDownload",
      "name": "ACTIVITY.tsv",
      "encodingFormat": "text/tab-separated-values",
      "contentUrl": "https://www.ebi.ac.uk/metabolights/MTBLS1234/files/ACTIVITY.tsv"
    },
    {
      "@type": "DataDownload",
      "name": "mzML raw files",
      "encodingFormat": "application/mzML+xml",
      "contentUrl": "https://www.ebi.ac.uk/metabolights/MTBLS1234/files/"
    }
  ]
}
```

***NOTE:*** This profile maps to `ACTIVITY.tsv` and supports data deposition in MetaboLights, GNPS, or other public repositories alongside the ChEMBL submission files.

***

## 3. Ontologies

### 3.1 BioAssay Ontology (BAO)

**Required Assay Classifications:**

* **BAO:0000697** - metabolism assay
* **BAO:0002989** - biotransformation assay
* **BAO:0000522** - cell-based assay
* **BAO:0000698** - cell-free assay
* **BAO:0002856** - enzymatic assay

**Assay Format:**

* **BAO:0000219** - single concentration response
* **BAO:0000357** - dose-response assay
* **BAO:0000208** - time-course assay

**Detection Method:**

* **BAO:0000196** - mass spectrometry
* **BAO:0000315** - chromatography
* **BAO:0000217** - spectrophotometry

### 3.2 Gene Ontology (GO) - Biological Process

**Biotransformation Processes:**

* **GO:0006805** - xenobiotic metabolic process
* **GO:0006082** - organic acid metabolic process
* **GO:0042737** - drug catabolic process
* **GO:0018894** - dibenzo-p-dioxin metabolic process
* **GO:0006725** - cellular aromatic compound metabolic process

**Molecular Functions:**

* **GO:0016491** - oxidoreductase activity
* **GO:0016740** - transferase activity
* **GO:0016787** - hydrolase activity
* **GO:0016829** - lyase activity
* **GO:0016853** - isomerase activity
* **GO:0016874** - ligase activity

### 3.3 Chemical Methods Ontology (CHMO)

**Analytical Techniques:**

* **CHMO:0000470** - liquid chromatography-mass spectrometry
* **CHMO:0000497** - gas chromatography-mass spectrometry
* **CHMO:0000591** - nuclear magnetic resonance spectroscopy
* **CHMO:0000228** - high performance liquid chromatography
* **CHMO:0000830** - ultra performance liquid chromatography

### 3.4 Reaction Types Ontology

**Common Biotransformation Reactions:**

| Reaction Type   | Description                   | EC Class    |
| --------------- | ----------------------------- | ----------- |
| Hydroxylation   | Addition of -OH group         | EC 1.14.x.x |
| Reduction       | Addition of H or removal of O | EC 1.x.x.x  |
| Oxidation       | Removal of H or addition of O | EC 1.x.x.x  |
| Hydrolysis      | Cleavage by water addition    | EC 3.x.x.x  |
| Decarboxylation | Loss of CO₂                   | EC 4.1.1.x  |
| Deamination     | Loss of NH₂                   | EC 3.5.x.x  |
| N-reduction     | Reduction of nitrogen groups  | EC 1.7.x.x  |
| Azo reduction   | Cleavage of N=N bonds         | EC 1.7.1.17 |
| Demethylation   | Loss of CH₃ group             | EC 1.14.x.x |
| Dehalogenation  | Loss of halogen               | EC 3.8.x.x  |
| Conjugation     | Addition of larger moiety     | EC 2.x.x.x  |
| Acetylation     | Addition of acetyl group      | EC 2.3.1.x  |
| Glucuronidation | Addition of glucuronic acid   | EC 2.4.1.17 |
| Sulfation       | Addition of sulfate           | EC 2.8.2.x  |

***

## 4. Assay Design and Experimental Setup

### 4.1 Assay Type Classification

**Level 1: Biological System**

* `whole cell assay` - Intact living cells
* `cell lysate assay` - Disrupted cells with enzymes
* `purified enzyme assay` - Isolated single enzyme
* `tissue/organ assay` - Ex vivo tissue preparation
* `community assay` - Mixed microbial population
* `in vivo assay` - Whole organism (e.g., animal model)

**Level 2: Experimental Format**

* `endpoint assay` - Single timepoint measurement
* `time-course assay` - Multiple timepoints
* `dose-response assay` - Multiple substrate concentrations
* `screening assay` - High-throughput format
* `mechanistic assay` - Detailed pathway elucidation

### 4.2 Required Assay Information

#### 4.2.1 Assay Identifier (AIDX)

Format: `A{number}_{OrganismAbbrev}_{Strain}_{Condition}`

**Examples:**

```
A001_Ecoli_K12_aerobic
A002_Btheta_VPI5482_anaerobic
A003_Csporogenes_ATCC15579_anaerobic
```

#### 4.2.2 Assay Description

**Required Components:**

1. **Biological system:** Whole cells, lysate, enzyme
2. **Substrate:** Xenobiotic compound name
3. **Organism:** Scientific name and strain
4. **Key conditions:** Aerobic/anaerobic, temperature
5. **Measurement:** What is being quantified

**Example:**

```
"Biotransformation of ibuprofen by Escherichia coli K-12 MG1655 
whole cells under anaerobic conditions at 37°C, measuring substrate 
depletion and metabolite formation by LC-MS/MS over 24 hours"
```

### 4.3 Substrate Preparation

**Required Information:**

| Parameter             | Required | Description                         |
| --------------------- | -------- | ----------------------------------- |
| Stock concentration   | Yes      | Concentration of stock solution     |
| Stock solvent         | Yes      | Solvent used (DMSO, ethanol, water) |
| Solvent percentage    | Yes      | Final % in assay (keep <1% organic) |
| Storage conditions    | Yes      | Temperature, light protection       |
| Working concentration | Yes      | Final concentration in assay        |
| Preparation method    | Yes      | Dissolution, sonication, etc.       |

**Example:**

```yaml
substrate_preparation:
  compound: "Ibuprofen"
  stock_solution:
    concentration: "100 mM"
    solvent: "DMSO"
    preparation: "dissolved with vortexing"
    storage: "-20°C, protected from light"
  working_solution:
    final_concentration: "100 µM"
    dilution_factor: "1:1000"
    solvent_percentage: "0.1% DMSO"
    preparation: "diluted in sterile medium"
```

### 4.4 Cell/Enzyme Preparation

**For Whole Cell Assays:**

```yaml
cell_preparation:
  culture_source: "overnight culture in BHI"
  harvest_method: "centrifugation 5000 × g, 10 min, 4°C"
  washing:
    buffer: "sterile PBS, pH 7.4"
    wash_cycles: 2
  resuspension:
    buffer: "fresh BHI medium"
    cell_density: "OD₆₀₀ = 1.0 (normalized)"
    cell_count: "~1 × 10⁹ CFU/mL"
  viability_check: ">95% viable (live/dead staining)"
```

**For Cell Lysate Assays:**

```yaml
lysate_preparation:
  cell_harvest: "as above"
  lysis_method: "sonication (3 × 30 s, on ice)"
  lysis_buffer: "50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂"
  centrifugation: "15000 × g, 20 min, 4°C"
  lysate_fraction: "supernatant (cytoplasmic fraction)"
  protein_concentration: "5 mg/mL (Bradford assay)"
  storage: "aliquots at -80°C, single use"
```

**For Purified Enzyme Assays:**

```yaml
enzyme_preparation:
  enzyme_source: "recombinant, expressed in E. coli"
  purification_method: "His-tag affinity chromatography"
  purity: ">95% (SDS-PAGE)"
  specific_activity: "12.5 U/mg"
  concentration: "0.5 mg/mL"
  storage_buffer: "50 mM Tris-HCl, pH 7.5, 20% glycerol"
  storage: "-80°C, avoid freeze-thaw"
```

### 4.5 Reaction Setup

**Standard Reaction Components:**

```yaml
reaction_mixture:
  total_volume: "1.0 mL"
  
  components:
    - name: "Cell suspension or lysate"
      volume: "900 µL"
      final_concentration: "OD₆₀₀ 1.0 or 5 mg/mL protein"
    
    - name: "Substrate (from stock)"
      volume: "1 µL"
      final_concentration: "100 µM"
    
    - name: "Cofactors (if needed)"
      components:
        - "NADH: 1 mM"
        - "NADPH: 1 mM"
        - "FAD: 50 µM"
    
    - name: "Solvent control"
      volume: "1 µL DMSO"
      purpose: "vehicle control"

  incubation:
    temperature: "37°C"
    atmosphere: "anaerobic (chamber or sealed tubes)"
    shaking: "100 rpm (or static)"
    duration: "0, 1, 3, 6, 12, 24 hours"
    
  controls:
    - "Negative control: heat-killed cells + substrate"
    - "Substrate control: substrate + medium only"
    - "Cell control: cells + solvent only"
```

### 4.6 Sampling and Quenching

**Required Information:**

```yaml
sampling_procedure:
  timepoints: [0, 1, 3, 6, 12, 24]  # hours
  sample_volume: "100 µL per timepoint"
  
  quenching_method:
    method: "acidification"
    reagent: "add 100 µL ice-cold acetonitrile with 0.1% formic acid"
    temperature: "on ice immediately"
    
  sample_processing:
    centrifugation: "15000 × g, 10 min, 4°C"
    supernatant_collection: "150 µL"
    storage: "-80°C until analysis"
    stability: "stable for 6 months"
    
  internal_standard:
    compound: "13C6-ibuprofen"
    concentration: "10 µM final"
    addition_timing: "at quenching step"
```

***

## 5. Analytical Methods and Detection

### 5.1 LC-MS/MS Method

**Required Method Parameters:**

```yaml
lcms_method:
  instrument:
    lc_system: "Agilent 1290 Infinity II UHPLC"
    ms_system: "Thermo Q Exactive Plus Orbitrap"
    
  chromatography:
    column: "Waters Acquity UPLC BEH C18"
    dimensions: "2.1 × 100 mm, 1.7 µm"
    column_temperature: "40°C"
    injection_volume: "5 µL"
    flow_rate: "0.3 mL/min"
    
    mobile_phase:
      A: "water + 0.1% formic acid"
      B: "acetonitrile + 0.1% formic acid"
    
    gradient:
      - time: "0 min", B: "5%"
      - time: "1 min", B: "5%"
      - time: "8 min", B: "95%"
      - time: "10 min", B: "95%"
      - time: "10.1 min", B: "5%"
      - time: "12 min", B: "5%"
  
  mass_spectrometry:
    ionization: "ESI negative mode"
    scan_type: "Full MS + dd-MS²"
    resolution: "70,000 FWHM (MS1), 17,500 (MS2)"
    mass_range: "50-1000 m/z"
    agc_target: "3e6"
    max_it: "100 ms"
    
    ms2_parameters:
      collision_energy: "stepped NCE 20, 40, 60"
      isolation_window: "1.0 m/z"
      
  data_acquisition:
    software: "Xcalibur 4.1"
    data_format: "mzML (centroid)"
```

### 5.2 Data Processing

**Required Information:**

```yaml
data_processing:
  peak_detection:
    software: "MZmine 2.53"
    mass_tolerance: "5 ppm"
    rt_tolerance: "0.1 min"
    min_peak_height: "1e4"
    
  peak_integration:
    method: "automated with manual review"
    baseline_correction: "local minimum"
    smoothing: "Savitzky-Golay (5 points)"
    
  compound_identification:
    level: "MSI Level 2"
    criteria:
      - "Accurate mass match (<5 ppm)"
      - "MS² spectral match (similarity >0.7)"
      - "Retention time match (±0.2 min)"
    reference_databases:
      - "MassBank"
      - "HMDB"
      - "GNPS"
      
  quantification:
    method: "external standard curve"
    calibration_range: "0.1-100 µM"
    calibration_points: 8
    curve_fit: "linear, 1/x weighting"
    r_squared: ">0.995"
    loq: "0.5 µM"
    lod: "0.1 µM"
```

### 5.3 Quality Control

**Required QC Measures:**

```yaml
quality_control:
  system_suitability:
    - "Column pressure: within specifications"
    - "Peak shape: tailing factor <1.5"
    - "Retention time stability: ±0.1 min"
    - "Mass accuracy: <5 ppm"
    
  blanks:
    - type: "Extraction blank"
      frequency: "every batch"
      acceptance: "no interfering peaks"
    - type: "Injection blank"
      frequency: "between samples"
      acceptance: "carryover <1%"
      
  standards:
    - type: "Calibration standard"
      frequency: "each batch"
      acceptance: "within 85-115% of expected"
    - type: "Quality control standard"
      frequency: "every 10 samples"
      acceptance: "within 80-120%, CV <20%"
      
  internal_standard:
    recovery: "70-130%"
    cv: "<20%"
```

***

## 6. Activity Measurements and Outcomes

### 6.1 Activity Types

**Primary Activities:**

| Activity Type           | Description              | Units    | ChEMBL Type |
| ----------------------- | ------------------------ | -------- | ----------- |
| Substrate depletion     | % of substrate remaining | % or µM  | Inhibition  |
| Product formation       | Amount of product formed | µM       | Product     |
| Transformation rate     | Rate of conversion       | µM/hr    | Rate        |
| Half-life               | Time to 50% conversion   | hours    | T50         |
| Biotransformation index | Ratio product/substrate  | unitless | Ratio       |

**Qualitative Activities:**

* `Substrate` - Compound consumed
* `Product` - Compound formed
* `No activity` - No transformation detected
* `Partial transformation` - Incomplete conversion
* `Complete transformation` - >95% conversion

### 6.2 Quantitative Measurements

**Required for Each Activity:**

```yaml
activity_measurement:
  cidx: "C001"
  aidx: "A001_Ecoli_K12"
  ridx: "REF_001"
  
  # Primary measurement
  type: "Biotransformation"
  action_type: "SUBSTRATE"
  
  substrate_depletion:
    initial_concentration: 100  # µM
    final_concentration: 15     # µM
    depletion_percentage: 85    # %
    relation: "="
    value: 85
    units: "%"
    sd_plus: 5.2
    sd_minus: 5.2
    
  # Transformation rate
  transformation_rate:
    value: 3.5
    units: "µM/hour"
    relation: "="
    calculation: "linear regression (0-24h)"
    r_squared: 0.98
    
  # Product formation
  product_formation:
    cidx: "P001"
    concentration: 72  # µM
    recovery: 85       # % of depleted substrate
    
  # Statistical parameters
  replicates:
    biological: 3
    technical: 2
  statistical_test: "one-way ANOVA"
  p_value: 0.003
  significance: "p < 0.01"
```

### 6.3 Dose-Response Data

**For Multiple Concentrations:**

```yaml
dose_response:
  substrate_concentrations: [1, 5, 10, 25, 50, 100, 200, 500]  # µM
  
  results:
    - concentration: 1
      activity: 10
      cv: 12
    - concentration: 5
      activity: 35
      cv: 8
    - concentration: 10
      activity: 55
      cv: 6
    # ... additional points
    
  curve_fitting:
    model: "Michaelis-Menten"
    km: 25.3  # µM
    vmax: 450  # nmol/min/mg protein
    hill_coefficient: 1.0
    r_squared: 0.96
```

### 6.4 Time-Course Data

**For Kinetic Studies:**

```yaml
time_course:
  timepoints: [0, 1, 3, 6, 12, 24, 48]  # hours
  
  substrate_profile:
    - time: 0
      concentration: 100.0
      sd: 0.0
    - time: 1
      concentration: 95.2
      sd: 3.1
    - time: 3
      concentration: 85.7
      sd: 4.5
    - time: 6
      concentration: 68.3
      sd: 5.2
    - time: 12
      concentration: 42.1
      sd: 6.8
    - time: 24
      concentration: 15.4
      sd: 4.2
    - time: 48
      concentration: 2.1
      sd: 0.8
      
  product_profile:
    - time: 0
      concentration: 0.0
    - time: 1
      concentration: 3.8
    # ... matching timepoints
    
  kinetics:
    rate_constant: 0.073  # h⁻¹
    half_life: 9.5        # hours
    model: "first-order decay"
```

***

## 7. Product Characterization

### 7.1 Metabolite Identification

**MSI Confidence Levels:**

**Level 1 - Identified Compounds:**

```yaml
metabolite:
  metabolite_id: "M001"
  identification_level: "Level 1"
  compound_name: "Hydroxy-ibuprofen"
  evidence:
    - "Authentic standard comparison"
    - "Retention time match (±0.05 min)"
    - "Accurate mass match (Δ <2 ppm)"
    - "MS² spectral match (similarity >0.95)"
  confidence: "Confirmed"
```

**Level 2 - Putatively Annotated:**

```yaml
metabolite:
  metabolite_id: "M002"
  identification_level: "Level 2"
  putative_name: "Carboxy-ibuprofen"
  evidence:
    - "Accurate mass (Δ <5 ppm)"
    - "MS² library match (similarity 0.85)"
    - "Expected biotransformation product"
  confidence: "Probable"
```

**Level 3 - Putatively Characterized:**

```yaml
metabolite:
  metabolite_id: "M003"
  identification_level: "Level 3"
  chemical_class: "Hydroxylated aromatic compound"
  evidence:
    - "Accurate mass indicates +16 Da (hydroxylation)"
    - "MS² fragmentation consistent with aromatic hydroxylation"
  confidence: "Tentative"
```

**Level 4 - Unknown:**

```yaml
metabolite:
  metabolite_id: "M004"
  identification_level: "Level 4"
  description: "Unknown biotransformation product"
  observed_mass: 237.1234
  retention_time: 5.67
```

### 7.2 Structural Characterization

**Required for New Metabolites:**

```yaml
structure_elucidation:
  metabolite_id: "M001"
  
  mass_spectrometry:
    exact_mass: 222.1256
    molecular_formula: "C13H18O3"
    formula_error: "1.2 ppm"
    double_bond_equivalents: 5
    
    ms2_fragments:
      - mz: 177.0916
        formula: "C11H13O2"
        assignment: "[M-COOH]-"
      - mz: 161.0966
        formula: "C11H13O"
        assignment: "[M-COOH-O]-"
        
  nmr_spectroscopy:
    acquired: true
    1h_nmr: "500 MHz, CD3OD"
    13c_nmr: "125 MHz, CD3OD"
    2d_experiments: ["COSY", "HSQC", "HMBC"]
    
  proposed_structure:
    smiles: "CC(C)Cc1ccc(cc1)C(C)(O)C(=O)O"
    inchi_key: "NEWMETABOLITE-UHFFFAOYSA-N"
    transformation_site: "tertiary carbon hydroxylation"
```

### 7.3 Transformation Pathway

**Pathway Documentation:**

```yaml
transformation_pathway:
  pathway_id: "PATH_001"
  substrate:
    cidx: "C001"
    name: "Ibuprofen"
    smiles: "CC(C)Cc1ccc(cc1)C(C)C(=O)O"
    
  steps:
    - step: 1
      reaction_type: "hydroxylation"
      enzyme: "cytochrome P450-like"
      product:
        metabolite_id: "M001"
        name: "Hydroxy-ibuprofen"
        smiles: "CC(C)Cc1ccc(cc1)C(C)(O)C(=O)O"
        
    - step: 2
      reaction_type: "oxidation"
      enzyme: "alcohol dehydrogenase"
      product:
        metabolite_id: "M002"
        name: "Carboxy-ibuprofen"
        smiles: "CC(C)Cc1ccc(cc1)C(=O)C(=O)O"
        
  branching_pathways:
    - branch: "A"
      steps: [1, 2]
      major: true
      percentage: 75
    - branch: "B"
      steps: [3]
      major: false
      percentage: 25
```

***

## 8. Data Formats - ACTIVITY.tsv

The MIX-MB(B) activity data lives in two formats: the **Template.xlsx Activity sheet** (the primary submitter input) and **ChEMBL ACTIVITY.tsv** (the pipeline output). The template is a superset of the ChEMBL format, adding metabolite detection, annotation, kinetic, and reaction-type columns as MIX-MB extensions.

***

### 8.1 MIX-MB(B) Activity Template (Template.xlsx — Activity Sheet)

This is the **primary submission format**. Submitters fill this sheet; the BioXend pipeline converts it to ChEMBL-ready files.

| #  | Column                        | Required | Type    | Description                                                                                                                                |
| -- | ----------------------------- | -------- | ------- | ------------------------------------------------------------------------------------------------------------------------------------------ |
| 1  | `Chemical_identifier`         | Yes\*    | String  | CIDX — auto-filled by pipeline. If filling manually, use the same identifier as in `COMPOUND_RECORD.tsv`.                                  |
| 2  | `Common_Name`                 | Yes      | String  | Common name of the xenobiotic (drug, pesticide, etc.) — must match the compound entry.                                                     |
| 3  | `SMILES`                      | Yes      | String  | Canonical SMILES of the parent compound — must match `COMPOUND_RECORD.tsv`.                                                                |
| 4  | `ASSAY_identifier`            | Yes      | String  | AIDX — must match a valid record in `ASSAY.tsv`. Use the vocabulary defined in the Assay sheet.                                            |
| 5  | `TEXT_VALUE`                  | Cond.    | String  | Qualitative activity result from controlled vocabulary (see 8.3). Leave empty when reporting a `VALUE`.                                    |
| 6  | `VALUE`                       | Cond.    | Numeric | Numerical measurement (e.g., % depletion, µM product). Leave empty when using `TEXT_VALUE`.                                                |
| 7  | `RELATION`                    | Cond.    | String  | Relationship operator for `VALUE`: `=`, `<`, `>`, `~`, `<=`, `>=`. Required when `VALUE` is provided.                                      |
| 8  | `UPPER_VALUE`                 | No       | Numeric | Upper bound of a range; `VALUE` is then the lower bound.                                                                                   |
| 9  | `UNITS`                       | Cond.    | String  | Unit of `VALUE` (e.g., `%`, `µM`, `µM/hour`, `h`). Required when `VALUE` is provided.                                                      |
| 10 | `ACTIVITY_COMMENT`            | **Yes**  | Text    | Free-text: thresholds, p-values, conditions, interpretation. **If left empty, BioXend will not annotate an activity value for this row.**  |
| 11 | `Metabolite_mz`               | No       | Numeric | Observed m/z of the detected biotransformation product (parent ion).                                                                       |
| 12 | `Metabolite_retention_time`   | No       | Text    | Retention time of the metabolite with unit (e.g., `2.17 min`).                                                                             |
| 13 | `Metabolite_annotation`       | No       | String  | Top annotation: SMILES, compound name, or ChemONT chemical class. Check a chemical database to determine the appropriate annotation level. |
| 14 | `Metabolite_annotation_level` | No       | Integer | MSI identification confidence: `1` (confirmed with authentic standard) to `5` (unknown). See Section 7 for full level definitions.         |
| 15 | `Kinetic_parameters`          | No       | Text    | Kinetic data — biotransformation assays only (e.g., Km, Vmax, rate constant k, half-life t½).                                              |
| 16 | `Reaction_type`               | No       | String  | Type of biotransformation reaction (controlled vocabulary in 8.3; EC numbers in Section 3.4).                                              |
| 17 | `Activity_type`               | Yes      | String  | Always `Biotransformation` for all entries in this standard.                                                                               |
| 18 | `ACTION_TYPE`                 | Cond.    | String  | Effect classification (see 8.3). **Leave empty if no confirmed activity — do not guess.**                                                  |

> \* `Chemical_identifier` is auto-filled by the BioXend pipeline; only supply manually when submitting without it.

***

### 8.2 ChEMBL ACTIVITY.tsv Mapping

The BioXend pipeline maps template columns to ChEMBL `ACTIVITY.tsv`. Columns 11–16 (metabolite, annotation, kinetics, reaction type) are MIX-MB extensions concatenated into `ACTIVITY_COMMENT`.

| ChEMBL Column      | Required | Template column         | Notes                                                                      |
| ------------------ | -------- | ----------------------- | -------------------------------------------------------------------------- |
| `CIDX`             | Yes      | 1 `Chemical_identifier` | Auto-generated or manually provided                                        |
| `AIDX`             | Yes      | 4 `ASSAY_identifier`    | Must match a row in `ASSAY.tsv`                                            |
| `RIDX`             | Yes      | Study-level             | Same across all activity rows in one study                                 |
| `TEXT_VALUE`       | Cond.    | 5 `TEXT_VALUE`          | Controlled vocabulary (see 8.3)                                            |
| `RELATION`         | Cond.    | 7 `RELATION`            | Required with `VALUE`                                                      |
| `VALUE`            | Cond.    | 6 `VALUE`               | Numerical measurement                                                      |
| `UPPER_VALUE`      | No       | 8 `UPPER_VALUE`         | For range measurements                                                     |
| `UNITS`            | Cond.    | 9 `UNITS`               | Required with `VALUE`                                                      |
| `ACTIVITY_COMMENT` | **Yes**  | 10 + cols 11–16         | Pipeline concatenates ACTIVITY\_COMMENT with metabolite and kinetic fields |
| `TYPE`             | Yes      | 17 `Activity_type`      | Always `Biotransformation`                                                 |
| `ACTION_TYPE`      | Cond.    | 18 `ACTION_TYPE`        | Null if no confirmed activity                                              |

***

### 8.3 Controlled Vocabularies for Activity Fields

**`TEXT_VALUE` — use one of these exact strings:**

| TEXT\_VALUE                | Meaning                               |
| -------------------------- | ------------------------------------- |
| `Compound metabolized`     | Parent compound consumed/depleted     |
| `Compound NOT metabolized` | No measurable transformation detected |
| `Product detected`         | Biotransformation product observed    |
| `Partial transformation`   | Incomplete conversion (< 95%)         |
| `Complete transformation`  | Full conversion (≥ 95%)               |

**`ACTION_TYPE` — effect classification:**

| ACTION\_TYPE  | Use when                                             |
| ------------- | ---------------------------------------------------- |
| `SUBSTRATE`   | The compound is consumed as a substrate              |
| `PRODUCT`     | The compound is a detected biotransformation product |
| `No Activity` | No transformation detected; leave `VALUE` empty      |
| `INHIBITION`  | The compound inhibits a microbial enzyme or process  |
| `STIMULATION` | The compound stimulates a microbial process          |

**`Reaction_type` — controlled vocabulary (see Section 3.4 for EC numbers):**

`hydroxylation` | `reduction` | `oxidation` | `hydrolysis` | `decarboxylation` | `deamination` | `N-reduction` | `azo_reduction` | `demethylation` | `dehalogenation` | `conjugation` | `acetylation` | `glucuronidation` | `sulfation`

***

### 8.4 Example Template Rows

**No biotransformation detected:**

| Col                  | Value                                                                           |
| -------------------- | ------------------------------------------------------------------------------- |
| Chemical\_identifier | HMM0089                                                                         |
| Common\_Name         | Diazepam                                                                        |
| SMILES               | C15H13ClN2O                                                                     |
| ASSAY\_identifier    | assay1                                                                          |
| TEXT\_VALUE          | Compound NOT metabolized                                                        |
| ACTIVITY\_COMMENT    | No biotransformation of Diazepam after 24h anaerobic incubation; background <2% |
| Activity\_type       | Biotransformation                                                               |
| ACTION\_TYPE         | No Activity                                                                     |

**Compound metabolised with putative metabolite (MSI Level 4):**

| Col                           | Value                                                                       |
| ----------------------------- | --------------------------------------------------------------------------- |
| Chemical\_identifier          | HMM0085                                                                     |
| Common\_Name                  | Diazepam                                                                    |
| ASSAY\_identifier             | assay3                                                                      |
| TEXT\_VALUE                   | Compound metabolized                                                        |
| ACTIVITY\_COMMENT             | Diazepam biotransformed to desmethyldiazepam; 85% conversion after 24h; n=3 |
| Metabolite\_mz                | 372.151                                                                     |
| Metabolite\_retention\_time   | 2.573 min                                                                   |
| Metabolite\_annotation        | Desmethyldiazepam                                                           |
| Metabolite\_annotation\_level | 4                                                                           |
| Reaction\_type                | hydrolysis                                                                  |
| Activity\_type                | Biotransformation                                                           |
| ACTION\_TYPE                  | SUBSTRATE                                                                   |

**Quantitative depletion with kinetics and confirmed product (MSI Level 2):**

| Col                           | Value                                                                                 |
| ----------------------------- | ------------------------------------------------------------------------------------- |
| Chemical\_identifier          | HMM0001                                                                               |
| Common\_Name                  | Ibuprofen                                                                             |
| SMILES                        | CC(C)Cc1ccc(cc1)C(C)C(=O)O                                                            |
| ASSAY\_identifier             | assay2                                                                                |
| TEXT\_VALUE                   | Compound metabolized                                                                  |
| VALUE                         | 85                                                                                    |
| RELATION                      | =                                                                                     |
| UNITS                         | %                                                                                     |
| ACTIVITY\_COMMENT             | 85% substrate depletion after 24h; SD ±5.2%; p<0.01 one-way ANOVA n=3; 37°C anaerobic |
| Metabolite\_mz                | 222.126                                                                               |
| Metabolite\_retention\_time   | 4.85 min                                                                              |
| Metabolite\_annotation        | CC(C)Cc1ccc(cc1)C(C)(O)C(=O)O                                                         |
| Metabolite\_annotation\_level | 2                                                                                     |
| Kinetic\_parameters           | t½=9.5h; k=0.073 h⁻¹                                                                  |
| Reaction\_type                | hydroxylation                                                                         |
| Activity\_type                | Biotransformation                                                                     |
| ACTION\_TYPE                  | SUBSTRATE                                                                             |

***

## 9. Controls and Validation

### 9.1 Required Controls

**Essential Controls:**

```yaml
experimental_controls:
  
  negative_controls:
    - type: "Heat-killed cells"
      preparation: "95°C for 20 min"
      purpose: "Confirm biological activity"
      expected: "No transformation"
      
    - type: "Medium only"
      composition: "Substrate + medium without cells"
      purpose: "Chemical stability"
      expected: "Substrate stable"
      
    - type: "Cells only"
      composition: "Cells + solvent without substrate"
      purpose: "Cell viability and background"
      expected: "No interfering peaks"
      
  positive_controls:
    - type: "Known substrate"
      compound: "Reference compound with known transformation"
      purpose: "System functionality"
      expected: "Expected transformation"
      
  internal_controls:
    - type: "Internal standard"
      compound: "Isotope-labeled substrate"
      purpose: "Extraction efficiency and quantification"
      expected: "Consistent recovery"
```

### 9.2 Quality Assurance

**Acceptance Criteria:**

```yaml
quality_assurance:
  
  assay_validity:
    - criterion: "Negative control"
      requirement: "<5% background transformation"
      measured: "2.1%"
      status: "PASS"
      
    - criterion: "Positive control"
      requirement: ">80% expected transformation"
      measured: "92%"
      status: "PASS"
      
    - criterion: "Internal standard recovery"
      requirement: "70-130%"
      measured: "105%"
      status: "PASS"
      
  reproducibility:
    - parameter: "Biological replicates (n=3)"
      cv: "8.5%"
      requirement: "<20%"
      status: "PASS"
      
    - parameter: "Technical replicates (n=2)"
      cv: "4.2%"
      requirement: "<15%"
      status: "PASS"
      
  analytical_performance:
    - parameter: "Mass accuracy"
      value: "2.3 ppm"
      requirement: "<5 ppm"
      status: "PASS"
      
    - parameter: "Retention time stability"
      value: "±0.08 min"
      requirement: "±0.2 min"
      status: "PASS"
```

***

## 10. Statistical Analysis

### 10.1 Required Statistical Information

```yaml
statistical_analysis:
  
  experimental_design:
    type: "Completely randomized design"
    independent_variable: "Substrate concentration"
    dependent_variable: "Biotransformation percentage"
    replicates:
      biological: 3
      technical: 2
      
  descriptive_statistics:
    mean: 85.3
    median: 86.1
    standard_deviation: 5.2
    standard_error: 3.0
    confidence_interval_95: "[79.4, 91.2]"
    
  hypothesis_testing:
    null_hypothesis: "No biotransformation occurs"
    alternative_hypothesis: "Biotransformation occurs"
    test: "one-sample t-test vs. 0% transformation"
    test_statistic: 28.4
    degrees_of_freedom: 2
    p_value: 0.0012
    significance_level: 0.05
    conclusion: "Reject null hypothesis (p < 0.05)"
    
  comparative_analysis:
    comparison: "Treatment vs. control"
    test: "two-sample t-test"
    p_value: 0.003
    effect_size_cohen_d: 2.45
    interpretation: "Large effect, highly significant"
```

### 10.2 Data Visualization Requirements

**Recommended Plots:**

* Time-course plot (concentration vs. time)
* Dose-response curve (activity vs. concentration)
* Bar chart with error bars (comparing conditions)
* Heatmap (multiple substrates × organisms)
* Pathway diagram (substrate → intermediates → products)

***

## 11. Data Quality Tiers

### Tier 1: Gold Standard (Publication-Ready)

* All Level A and B information complete
* Quantitative activity data with statistical analysis (n ≥ 3 biological replicates)
* Metabolite products identified at MSI Level 1 or 2
* Raw LC-MS data deposited in a public repository (MetaboLights, GNPS)
* Transformation pathway documented with proposed mechanism
* All required controls passed (negative, positive, internal standard)
* Mass accuracy ≤ 5 ppm; internal standard recovery 70–130%

### Tier 2: Silver Standard (Research-Grade)

* All Level A information complete
* Quantitative or semi-quantitative activity data (n ≥ 2 biological replicates)
* Metabolite products identified at MSI Level 2 or 3
* Negative and internal standard controls included
* Basic analytical quality control documented

### Tier 3: Bronze Standard (Preliminary)

* All Level A essential information (CIDX, AIDX, RIDX, TEXT\_VALUE, ACTION\_TYPE)
* Qualitative activity (substrate consumed / product detected / no activity)
* Single measurement acceptable for initial screening
* Suitable for large-scale screening studies where quantification is impractical

***

## 12. Example Complete Record

```yaml
# MIX-MB(B) Compliant Biotransformation Record

# Assay Identification
assay_id: "A001_Ecoli_K12_Ibuprofen"
assay_date: "2023-09-15"
assay_type: "whole cell biotransformation"
assay_format: "time-course with dose-response"

# Links to Other Standards
substrate_reference: "C001"  # from MIX-MB(X)
organism_reference: "ORG_001"  # from MIX-MB(M)
publication_reference: "REF_001"

# Assay Description
description: >
  Biotransformation of ibuprofen by Escherichia coli K-12 MG1655
  under anaerobic conditions. Whole cells were incubated with 
  substrate at 37°C for 24 hours. Substrate depletion and metabolite
  formation were quantified by LC-MS/MS.

# Experimental Setup
substrate:
  cidx: "C001"
  name: "Ibuprofen"
  chembl_id: "CHEMBL1201246"
  stock_concentration: "100 mM in DMSO"
  working_concentrations: [10, 25, 50, 100, 200]  # µM
  final_concentration_tested: 100  # µM
  solvent_percentage: 0.1  # % DMSO

organism:
  organism_id: "ORG_001"
  scientific_name: "Escherichia coli"
  ncbi_taxid: 511145
  strain: "K-12 substr. MG1655"

cell_preparation:
  culture_medium: "LB broth"
  growth_phase: "stationary phase"
  harvest_od600: 2.0
  washing: "2× PBS"
  resuspension_medium: "M9 minimal medium"
  final_od600: 1.0
  cell_concentration: "~8 × 10⁸ CFU/mL"

reaction_conditions:
  volume: "1.0 mL"
  temperature: "37°C"
  atmosphere: "anaerobic (chamber, <0.1% O₂)"
  shaking: "100 rpm"
  duration: "24 hours"
  sampling_times: [0, 3, 6, 12, 24]  # hours

controls:
  negative:
    - "Heat-killed cells (95°C, 20 min) + substrate"
    - "Medium only + substrate"
  positive:
    - "Known substrate transformation (aspirin)"
  internal_standard:
    - "13C6-ibuprofen, 10 µM"

# Analytical Method
analytical_method:
  platform: "LC-MS/MS"
  instrument: "Agilent 1290 UHPLC - Thermo Q Exactive Plus"
  
  chromatography:
    column: "Waters BEH C18, 2.1×100mm, 1.7µm"
    mobile_phase_A: "H₂O + 0.1% FA"
    mobile_phase_B: "ACN + 0.1% FA"
    flow_rate: "0.3 mL/min"
    gradient: "5-95% B in 8 min"
    
  mass_spectrometry:
    ionization: "ESI negative"
    resolution: "70,000"
    mass_range: "50-1000 m/z"
    fragmentation: "HCD, NCE 40"

# Results
activity_data:
  substrate_activity:
    cidx: "C001"
    aidx: "A001_Ecoli_K12_Ibuprofen"
    ridx: "REF_001"
    type: "Biotransformation"
    action_type: "SUBSTRATE"
    
    initial_concentration: 100  # µM
    final_concentration: 12    # µM
    depletion: 88              # %
    depletion_sd: 4.5          # %
    
    transformation_rate: 3.67  # µM/hour
    rate_sd: 0.31
    
    biological_replicates: 3
    technical_replicates: 2
    
    statistical_test: "one-way ANOVA"
    p_value: 0.0008
    significance: "p < 0.001"
    
  product_activity:
    cidx: "P001"
    metabolite_id: "M001"
    name: "Hydroxy-ibuprofen"
    identification_level: "Level 2"
    
    concentration: 78  # µM
    recovery: 88.6     # % of depleted substrate
    
    exact_mass: 222.1256
    retention_time: 4.85  # min
    ms2_match_score: 0.87

# Transformation Pathway
pathway:
  reaction_type: "reductive hydroxylation"
  enzyme_class: "EC 1.14.x.x (oxidoreductase)"
  cofactor_requirement: "NADPH-dependent"
  
  proposed_mechanism: >
    Cytochrome P450-like enzyme catalyzes hydroxylation at the
    tertiary carbon of the isobutyl side chain.

# Quality Control
qc_results:
  negative_control: "PASS - <2% background"
  positive_control: "PASS - 95% expected transformation"
  internal_standard_recovery: "102% (PASS: 70-130%)"
  biological_replicate_cv: "5.1% (PASS: <20%)"
  technical_replicate_cv: "2.8% (PASS: <15%)"
  mass_accuracy: "1.8 ppm (PASS: <5 ppm)"

# Data Quality
quality_tier: "Gold Standard"
data_completeness: "100% Level A, 95% Level B"
validation_status: "PASS"
raw_data_available: true
raw_data_repository: "MetaboLights"
raw_data_accession: "MTBLS1234"

# Contact
principal_investigator: "Dr. Jane Smith"
laboratory: "Microbial Metabolism Lab, University XYZ"
date_performed: "2023-09-15"
date_analyzed: "2023-09-20"
```

***

## 13. Integration with Other Standards

### 13.1 Complete Data Package

A complete biotransformation study submission includes:

```
Study Package
├── MIX-MB(X) - Xenobiotics
│   ├── COMPOUND_RECORD.tsv
│   ├── COMPOUND_CTAB.sdf
│   └── Chemical structures
│
├── MIX-MB(M) - Microbes  
│   ├── Organism metadata
│   ├── Growth conditions
│   └── Culture information
│
├── MIX-MB(B) - Biotransformation (THIS STANDARD)
│   ├── ASSAY.tsv
│   ├── ASSAY_PARAM.tsv
│   ├── ACTIVITY.tsv
│   └── Experimental details
│
├── REFERENCE.tsv
│   └── Publication metadata
│
└── Raw Data
    ├── LC-MS data (mzML)
    ├── NMR data (nmrML)
    └── Analysis scripts
```

### 13.2 Cross-Referencing

**Linking Strategy:**

```yaml
cross_references:
  substrate:
    cidx: "C001"
    from_standard: "MIX-MB(X)"
    file: "COMPOUND_RECORD.tsv"
    
  organism:
    organism_id: "ORG_001"
    ncbi_taxid: 511145
    from_standard: "MIX-MB(M)"
    
  assay:
    aidx: "A001"
    links_to: ["C001", "ORG_001"]
    from_standard: "MIX-MB(B)"
    file: "ASSAY.tsv"
    
  activity:
    links_assay: "A001"
    links_compound: "C001"
    from_standard: "MIX-MB(B)"
    file: "ACTIVITY.tsv"
```

***

## 14. Data Access and Repository Guidance

### 14.1 Repository and Standard Documents

The MIX-MB standard documents, submission template, and BioXend pipeline are openly available:

* **Standard documents:** [GitHub — zmahnoor14/BioXend](https://github.com/zmahnoor14/BioXend), under `Standards/`
* **Submission template:** `Standards/Templates/Template.xlsx` — the **Activity** sheet covers MIX-MB(B)
* **BioXend pipeline:** Nextflow workflow (`main.nf`) in the repository root

### 14.2 Files Generated by This Standard

MIX-MB(B) experimental data feed the activity and assay parameter files required for ChEMBL deposition. The BioXend pipeline produces:

| Output File       | Generator Script       | Content                                         |
| ----------------- | ---------------------- | ----------------------------------------------- |
| `ASSAY.tsv`       | `generate_assay.R`     | Assay descriptions per organism × condition     |
| `ASSAY_PARAM.tsv` | `generate_assay.R`     | Quantitative experimental parameters            |
| `ACTIVITY.tsv`    | `write_activity_tsv.R` | Compound–assay activity links with measurements |

These files are joined to compound records via CIDX (MIX-MB(X)) and organism records via AIDX (MIX-MB(M)).

### 14.3 Submitting to ChEMBL

MIX-MB(B) activity files are deposited to [ChEMBL](https://www.ebi.ac.uk/chembl/) as part of the complete six-file MIX-MB submission package. For deposition enquiries, contact **<chembl-help@ebi.ac.uk>**.

**Pre-submission checklist for activity data:**

* Every `ACTIVITY.tsv` row has a valid CIDX, AIDX, and RIDX
* `ACTION_TYPE` uses only controlled vocabulary terms (Section 8.3)
* `ACTIVITY_COMMENT` is non-empty for every row
* All controls (Section 9) are documented in `ASSAY_PARAM.tsv` or `ASSAY_DESCRIPTION`

### 14.4 Raw Data Repositories

Raw analytical data accompanying a MIX-MB(B) submission should be deposited alongside the ChEMBL files:

| Repository   | URL                                   | Data type                                     |
| ------------ | ------------------------------------- | --------------------------------------------- |
| MetaboLights | <https://www.ebi.ac.uk/metabolights/> | LC-MS raw data (.mzML), processed peak tables |
| GNPS         | <https://gnps.ucsd.edu/>              | MS/MS spectral data, molecular networks       |
| MassIVE      | <https://massive.ucsd.edu/>           | Raw MS data (alternative to MetaboLights)     |

Record the repository accession in `ACTIVITY_COMMENT` and in the Bioschemas Dataset `@id` field (Section 2.3).

***

## 15. Licence and Reuse

### 15.1 Standard Documents

The MIX-MB standard documents are released under the **Creative Commons Attribution 4.0 International (CC BY 4.0)** licence (<https://creativecommons.org/licenses/by/4.0/>).

You are free to share, adapt, and build upon this standard for any purpose, provided you give appropriate credit:

> Zulfiqar M. *et al.* MIX-MB: Minimum Information about Xenobiotics-Microbiome Biotransformation (v0.1.0). GitHub: <https://github.com/zmahnoor14/BioXend>

### 15.2 Pipeline and Code

The BioXend Nextflow pipeline and associated scripts are released under the **MIT Licence**. See `LICENSE` in the repository root for full terms.

### 15.3 Submitted Data

Data deposited using MIX-MB formats in public repositories are subject to that repository's terms:

| Repository     | Licence                                                          |
| -------------- | ---------------------------------------------------------------- |
| ChEMBL         | CC BY-SA 3.0 (<https://creativecommons.org/licenses/by-sa/3.0/>) |
| MetaboLights   | CC0 1.0 or CC BY 4.0 (depositor's choice at submission)          |
| GNPS / MassIVE | CC0 (public domain)                                              |

When reusing MIX-MB-formatted datasets, cite both the original study (via its RIDX/DOI) and the MIX-MB standard.

***

## 16. Provenance

Provenance records the origin, history, and chain of custody of biotransformation experimental data reported under MIX-MB(B). Complete provenance supports the **Reusable** principle of FAIR and enables independent reproducibility assessment.

### 16.1 Study-Level Provenance

Every activity record is anchored to a **Reference record** (RIDX) in `REFERENCE.tsv`:

| Field                      | Description                                             |
| -------------------------- | ------------------------------------------------------- |
| `RIDX`                     | Study-local identifier linking all six submission files |
| `DOI`                      | Digital Object Identifier of the source publication     |
| `TITLE`, `AUTHORS`, `YEAR` | Full bibliographic metadata                             |

The RIDX appears in every row of `ACTIVITY.tsv`, `ASSAY.tsv`, and `COMPOUND_RECORD.tsv`, making the publication the root provenance node for the entire data package.

### 16.2 Experimental Provenance

MIX-MB(B) traceability runs from raw measurement back to the study:

```
REFERENCE.tsv  (RIDX)
       │
       ├── COMPOUND_RECORD.tsv  (CIDX) ← chemical identity and source (MIX-MB(X))
       ├── ASSAY.tsv            (AIDX) ← organism, strain, and conditions (MIX-MB(M))
       └── ACTIVITY.tsv   (CIDX × AIDX × RIDX) ← measured outcome
```

Each activity row therefore carries the full experimental context via its three index keys.

### 16.3 Protocol and Method Provenance

Document the analytical and experimental methods in:

| Location                                    | What to record                                                    |
| ------------------------------------------- | ----------------------------------------------------------------- |
| Bioschemas LabProtocol record (Section 2.2) | Step-by-step protocol with reagents and instruments               |
| `ASSAY_PARAM.tsv`                           | Quantitative parameters (temperature, duration, cell density, pH) |
| `ASSAY_DESCRIPTION` in `ASSAY.tsv`          | Free-text assay description including key conditions              |
| `ACTIVITY_COMMENT`                          | Analytical method details, processing software, mass accuracy     |

**Minimum method provenance for Gold-tier submissions (Tier 1):**

* Instrument model and manufacturer
* Software versions used for data acquisition and processing (e.g., MZmine 2.53, Xcalibur 4.1)
* Spectral library used for metabolite annotation (e.g., MassBank, HMDB)

### 16.4 Raw Data Provenance

For each study, record:

* **MetaboLights / GNPS accession** — in `ACTIVITY_COMMENT` and in the Dataset Bioschemas `@id` (Section 2.3)
* **Raw data format** — mzML (preferred) or vendor format with conversion notes
* **Processing pipeline** — version and parameters used (e.g., MZmine feature detection settings)

### 16.5 Pipeline Provenance

When generating submission files with BioXend, record in `REFERENCE.tsv` or `ASSAY_DESCRIPTION`:

* **BioXend pipeline version** — from `versions/pipeline.txt`
* **Date of file generation** — ISO 8601 format (YYYY-MM-DD)

***

## 17. Version History

| Version | Date       | Changes                                         |
| ------- | ---------- | ----------------------------------------------- |
| 0.1.0   | 2026-02-05 | Initial draft: Core biotransformation standards |

***

## 18. References

1. ChEMBL Bioactivity Database: <https://www.ebi.ac.uk/chembl/>
2. BioAssay Ontology (BAO): <http://www.bioassayontology.org/>
3. MSI Metabolomics Standards: <https://metabolomicssociety.org/>
4. STRENDA Guidelines: <http://www.beilstein-strenda-db.org/>
5. Gene Ontology: <http://geneontology.org/>
6. Chemical Methods Ontology: <https://terminology.nfdi4chem.de/ts/ontologies/chmo>
7. MetaboLights: <https://www.ebi.ac.uk/metabolights/>
8. GNPS: <https://gnps.ucsd.edu/>
9. mzML Standard: <http://www.psidev.info/mzml>
10. FAIR Principles: <https://www.go-fair.org/fair-principles/>

***

## 19. Contact and Contributions

For questions, suggestions, or contributions to this standard, please contact:

* **Maintainer:** Mahnoor Zulfiqar
* **Institution:** NFDI4Microbiota
* **Email:** [Contact information](mailto:zmahnoor14@gmail.com)
* **Repository:** \[[GitHub repository URL](https://github.com/zmahnoor14/BioXend)]

This standard bridges MIX-MB(X) and MIX-MB(M) to provide comprehensive documentation of microbial xenobiotic biotransformation processes. It is a living document that will be updated based on community feedback and evolving best practices.

***


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