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すべてのプロトコル分野

Protein & Antibody Workflows — Bottom-up MS first child

タンパク質名を増やさず、実際の workflow で設計

最初の bounded child は定量済み互換タンパク質溶液から始まり、exact digestion source、1つの RP cleanup IFU、実施設の handoff を接続します。メーカー条件の変更は必ず明示します。

編集可能な開始プロトコル

1 bounded workflow

このプロトコルは何を行いますか？

定量済みprotein solutionをsourceに沿って変性・還元・アルキル化・消化・RP cleanupし、LC–MS/MS施設へ引き渡せる状態にします。

Workflowの範囲

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

適用範囲

FASP、S-Trap、SP3、iST、in-gel digestionは別workflowです。異なるメーカー条件は明示的なpersonalized変更としてのみ扱います。

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

公式ガイド 確認済み
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

目的と範囲

定量・matrix 互換性を確認した研究用 protein solution を peptide に変換し、施設別 pre-acquisition handoff を作成します。注入適合性や解析成功は保証しません。

必須 starting record

  • sample identity・material role・目的
  • 濃度・使用可能量・assay run provenance
  • 正確な buffer・additives・upstream history
  • reduction / alkylation state
  • exact digestion・cleanup・facility source

開始前確認

  • current matrix を完全に記録
  • exact assay kit compatibility を確認
  • 完了 chemistry の重複防止
  • protease と cleanup の product/catalog/revision を固定
  • 施設の dry/reconstituted endpoint を先に確認

判断・停止ルール

  • 未定量・unknown matrix は export lock
  • hidden precipitation/buffer exchange/detergent removal は別 route
  • FASP・S-Trap・SP3・iST・in-gel は内部 option ではない
  • cleanup product/format 変更時は source reset
  • facility instruction なしでは handoff 完了不可

別 child route

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

標準 workflow — universal 値ではなく順序と gate を標準化

  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

終了点

Pre-acquisition Peptide Handoff · Research Use Only。最終状態、保管、vial/label、source set、change ledger、facility instruction を記録します。

reference の使い方

reference は optional reading であり、選択 block の governing source です。Exact 値は当該 product/revision のみに適用します。

メーカー protocol を baseline とし、追加・削除・置換した条件を ADD / REMOVE / REPLACE で強調し、理由と local qualification を残してください。

使用前に自分のassay情報を入力してください

実行順序と合理的な開始条件を整理した下書きです。サンプルのprimer・probe配列は含めていません。必ず自分の標的配列を入力し、実際の酵素・kit・装置の説明書とassay検証データに合わせて条件を確定してください。

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.

条件: 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.

試薬: USER REQUIRED — exact assay kit and run record条件: 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.

条件: 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.

試薬: USER REQUIRED — selected protease product and official source条件: 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.

時間: USER REQUIRED — selected source value and locator温度: USER REQUIRED — selected source value and locator試薬: USER REQUIRED — selected source chemistry and preparation条件: 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.

時間: USER REQUIRED — exact source incubation and stop checkpoints温度: USER REQUIRED — exact source temperature試薬: USER REQUIRED — exact protease amount or ratio and stock provenance条件: 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.

条件: 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.

時間: USER REQUIRED — exact IFU step or endpoint試薬: USER REQUIRED — exact cleanup product and IFU solutions条件: 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.

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