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전체 프로토콜 분야

Protein & Antibody Workflows — Bottom-up MS first child

단백질을 이름별로 늘리지 않고, 실제 workflow로 설계

첫 bounded child는 정량된 호환 단백질 용액에서 시작해 exact digestion source, 한 RP cleanup IFU, 실제 분석시설 인계조건을 연결합니다. 제조사 protocol을 바꾸거나 섞으면 차이를 반드시 기록합니다.

편집 가능한 시작 프로토콜

1 bounded workflow

이 프로토콜은 무엇을 하나요?

정량된 compatible protein solution을 입력으로 받아 source에 맞춘 변성·환원·알킬화·소화와 RP cleanup을 거쳐 LC–MS/MS 시설 인계 상태를 만듭니다.

워크플로 범위

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

적용 경계

FASP, S-Trap, SP3, iST, in-gel digestion을 이 절차와 혼합하지 않습니다. 서로 다른 제조사 조건은 personalized 변경으로만 명시합니다.

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

공식 가이드 검토됨
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

목적과 범위

정량과 matrix 호환성이 확인된 research protein solution을 peptide로 변환해 시설별 pre-acquisition handoff를 만드는 제품입니다. 분석 성공이나 instrument 주입 적합성을 인증하지 않습니다.

시작 물질에 반드시 필요한 것

  • sample identity·material role·연구 목적
  • 농도·가용 부피·assay kit/run provenance
  • 정확한 buffer/additives와 upstream history
  • reduction/alkylation state
  • digestion·cleanup·facility의 exact source

시작 전 확인

  • 현재 matrix를 성분과 농도까지 기록
  • 실제 assay kit의 compatibility 확인
  • 완료 chemistry 중복 방지
  • protease와 RP cleanup의 product/catalog/revision 고정
  • 시설이 받을 dry/reconstituted state 선확인

결정과 중단 기준

  • 미정량·미상 matrix는 export 중단
  • 숨은 precipitation/buffer exchange/detergent removal은 다른 workflow로 이동
  • FASP·S-Trap·SP3·iST·in-gel은 내부 옵션이 아님
  • cleanup product/format 변경 시 source 재선택
  • 시설 instruction이 없으면 handoff 완료 금지

별 child로 분리되는 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

표준 workflow — 값이 아니라 순서와 gate를 표준화

  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

종료점

Pre-acquisition Peptide Handoff · Research Use Only. dry/reconstituted state, storage, vial/label, source set, change ledger, facility instruction을 남깁니다.

reference 사용법

reference는 optional reading이면서 선택한 block의 governing source입니다. Exact 값은 해당 product와 revision에만 적용됩니다.

제조사 protocol을 그대로 출발점으로 사용하되, 추가·삭제·교체한 모든 조건을 ADD / REMOVE / REPLACE로 강조하고 이유와 local qualification을 남기세요.

먼저 내 assay 정보를 채워 주세요

실행 순서와 합리적인 시작 조건을 정리한 초안입니다. 샘플 primer·probe 서열은 넣지 않았습니다. 반드시 자신의 표적 서열을 입력하고, 실제 효소·kit·장비 설명서와 assay 검증 결과에 맞게 조건을 확정하세요.

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.

조건: 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.

시약: USER REQUIRED — exact assay kit and run record조건: 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.

조건: 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.

시약: USER REQUIRED — selected protease product and official source조건: 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.

시간: USER REQUIRED — selected source value and locator온도: USER REQUIRED — selected source value and locator시약: USER REQUIRED — selected source chemistry and preparation조건: 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.

시간: USER REQUIRED — exact source incubation and stop checkpoints온도: USER REQUIRED — exact source temperature시약: USER REQUIRED — exact protease amount or ratio and stock provenance조건: 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.

조건: 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.

시간: USER REQUIRED — exact IFU step or endpoint시약: USER REQUIRED — exact cleanup product and IFU solutions조건: 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.

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