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Protein & Antibody Workflows — Bottom-up MS first child

Organize Protein by workflows, not by an endless list of names

The first bounded child starts with a quantified compatible protein solution and connects one exact digestion source, one RP cleanup IFU, and the actual facility handoff. Every manufacturer change must be explicit.

Editable starter protocols

1 bounded workflow

What does this protocol do?

It takes a quantified compatible protein solution through source-aligned denaturation, reduction, alkylation, digestion, RP cleanup, and LC–MS/MS facility handoff.

Workflow coverage

  • • B. Bottom-up MS · in-solution + RP cleanup

Scope boundary

FASP, S-Trap, SP3, iST, and in-gel digestion remain separate workflows. Conditions from different manufacturers are recorded only as explicit personalized changes.

PRT-PROTEIN-BOTTOMUP-NORM-001B. Bottom-up MS · in-solution + RP cleanup

Source-Governed In-Solution Protein Digestion and RP Peptide Cleanup

Protein & Antibody Workflows > Bottom-up MS Proteomics · PRT-PROTEIN-B1-P1A-P1B-20260828

Official guidance reviewed
QUANTIFIED_COMPATIBLE_SOLUTION_ONLYFOUR_SOURCE_BLOCKS_REQUIREDSOURCE_GOVERNED_COMPOSEDCONNECT_HANDOFF_CONTRACT_REQUIREDONE_EXACT_RP_CLEANUP_IFUADD_REMOVE_REPLACE_FOR_PERSONALIZATIONNO_UNIVERSAL_NUMERIC_DEFAULTSPRE_ACQUISITION_HANDOFF_RESEARCH_ONLY

PROTEIN WORKFLOW FOUNDATION

Purpose and scope

Convert a quantified, matrix-compatible research protein solution into a facility-specific pre-acquisition peptide handoff. This product does not certify injection suitability or analytical success.

Required starting record

  • sample identity, material role, and purpose
  • concentration, available volume, and assay run provenance
  • exact buffer, additives, and upstream history
  • reduction and alkylation state
  • exact digestion, cleanup, and facility sources

Before starting

  • record the complete current matrix
  • check the exact assay kit compatibility
  • prevent duplicate completed chemistry
  • lock protease and cleanup product/catalog/revision
  • confirm the facility’s dry or reconstituted endpoint first

Decision and stop rules

  • unquantified or unknown-matrix input locks export
  • hidden precipitation, buffer exchange, or detergent removal routes elsewhere
  • FASP, S-Trap, SP3, iST, and in-gel are not internal options
  • changing cleanup product or format resets the source
  • no facility instruction means no completed handoff

Separate child routes

  • upstream extraction or precipitation
  • FASP filter reactor
  • S-Trap suspension trap
  • SP3/PAC bead workflow
  • iST or integrated kit
  • in-gel digestion
  • labeling, fractionation, or PTM enrichment

Standard workflow — standardize order and gates, not universal values

  1. 0. Experiment Definition & Material Role
  2. 1. Sample State and Quantification Provenance
  3. 2. Current Matrix / Compatibility Review
  4. 3. In-Solution Digestion Source Strategy
  5. 4. Reduction / Alkylation State
  6. 5. Ordered Protease Digestion and Stop
  7. 6. Digestion-to-Cleanup Handoff Contract
  8. 7. One Exact RP Cleanup IFU
  9. 8. Dry/Reconstituted Pre-acquisition Handoff

Terminal handoff

Pre-acquisition Peptide Handoff · Research Use Only. Record final state, storage, vial/label, adopted sources, changes, and the facility instruction.

How to use references

A reference is optional reading and the governing record for its selected block. Exact values apply only to that product and revision.

Use a manufacturer protocol as the baseline, then highlight every added, removed, or replaced condition with a rationale and local qualification.

Add your assay information before use

This draft provides an ordered workflow and rational starting conditions. It intentionally contains no sample primer or probe sequence. Enter your target-specific oligonucleotides and finalize every condition against the actual enzyme, kit, instrument instructions, and assay-validation data.

1

0. DEFINE THE EXPERIMENT AND MATERIAL ROLE

Identify the sample, biological source or material, research purpose, analyte role, and intended downstream analysis. Confirm that this child starts from an already quantified protein solution rather than cells, tissue, pellet, gel band, or an unspecified lysate.

Condition: Route incompatible starting states to a planned extraction, precipitation, FASP, S-Trap, SP3, iST, or in-gel child instead of hiding upstream processing.
2

1. RECORD SAMPLE STATE AND QUANTIFICATION PROVENANCE

Record measured concentration, available volume, calculated amount, exact assay kit and lot, standard/blank/run identifier, result date, and the source locator used to interpret the assay. Keep measured, user-entered, and derived values distinguishable.

Reagent: USER REQUIRED — exact assay kit and run recordCondition: No unquantified input and no universal conversion between assay chemistries.
3

2. REVIEW THE CURRENT MATRIX AND ROUTE COMPATIBILITY

List the complete buffer and additive composition, concentration where known, pH, detergent or chaotrope, salts, inhibitors, carriers, and upstream clarification or matrix-change history. Compare the actual matrix separately against the assay, digestion, cleanup, and facility source blocks.

Condition: A simple dilution may change concentration and volume while preserving the matrix identity. Precipitation, buffer exchange, detergent removal, or pH correction is a different operation and routes out of B1.
4

3. ADOPT ONE EXACT IN-SOLUTION DIGESTION SOURCE

Select the actual protease product, catalog, lot, official URL, revision, and applicable in-solution section. Transcribe the ordered source conditions into the immutable snapshot, then create a separate working copy. Do not average or mix protease protocols.

Reagent: USER REQUIRED — selected protease product and official sourceCondition: The curated Promega TM390 profile is available as a source block, but it applies only when that exact product is used.
5

4. DECLARE REDUCTION AND ALKYLATION STATE

For each chemistry operation, record execute, skip already complete, or route out. When execution is required, use the adopted source identity, order, reagent preparation, endpoint, and safety instructions. Preserve already completed chemistry in the handoff record.

Duration: USER REQUIRED — selected source value and locatorTemperature: USER REQUIRED — selected source value and locatorReagent: USER REQUIRED — selected source chemistry and preparationCondition: Skipping an already completed operation prevents duplication and is not counted as a personalized protocol change.
6

5. EXECUTE ORDERED PROTEASE DIGESTION AND SOURCE-DEFINED STOP

Calculate sample and protease additions only from measured/user values and the selected source instruction. Preserve enzyme order, source-defined conditions, incubation endpoint, and stop or acidification operation. Record deviations in the change ledger.

Duration: USER REQUIRED — exact source incubation and stop checkpointsTemperature: USER REQUIRED — exact source temperatureReagent: USER REQUIRED — exact protease amount or ratio and stock provenanceCondition: No built-in enzyme ratio, time, temperature, or digestion-completeness claim.
7

6. PASS THE DIGESTION-TO-CLEANUP CONNECT CONTRACT

Describe the peptide mixture after stop: material role, total volume, matrix composition, pH or acidification state, organic-solvent fraction, estimated load provenance, and any visible issue. Confirm that these outputs satisfy the exact cleanup input requirements.

Condition: If the cleanup IFU does not accept the recorded mixture, stop and revise the route. Do not silently invent a correction step.
8

7. APPLY ONE EXACT RP PEPTIDE CLEANUP IFU

Adopt exactly one RP product snapshot and follow its source-governed condition, load, wash, and elution sequence. Record product format, catalog, lot, revision, source locator, actual load, collection identity, and any split or deviation.

Duration: USER REQUIRED — exact IFU step or endpointReagent: USER REQUIRED — exact cleanup product and IFU solutionsCondition: The curated Pierce 84850 profile applies only to its 20 uL spin-tip format. It does not predict recovery or authorize another C18 format.
9

8. CLOSE THE PRE-ACQUISITION PEPTIDE HANDOFF

Record whether the peptide is dry or reconstituted, the actual amount/volume provenance, solution if applicable, storage, transport, vial and label, sample sheet, organism and enzyme metadata, receiving facility instruction/version, deviations, and unresolved items.

Condition: Output is Pre-acquisition Peptide Handoff · Research Use Only. It does not certify instrument-injection suitability, recovery, sensitivity, completeness, or analytical success.