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ANALYTICAL TECHNOLOGY

Standardize earlier in the protein workflow.

A full-length SIL protein can enter a bottom-up LC-MS/MS method before relevant processing and digestion. The method determines what variation it can reflect.

THE PRINCIPLE

Shared steps, distinct masses.

Light analyte and labeled protein can proceed through the same later steps. Corresponding peptides are then measured by LC-MS/MS.

  1. 01
    SampleDefine matrix and target
  2. 02
    Add full-length SIL proteinBefore relevant processing
  3. 03
    Process / extractMethod-specific steps
  4. 04
    DigestFor bottom-up methods
  5. 05
    LCSeparate peptides
  6. 06
    MS detectionMeasure light / heavy
  7. 07
    QuantificationApply method calibration

A standard only encounters steps after its addition. Intact-protein workflows omit digestion.

METHOD CHOICE

Where protein and peptide standards differ.

General methodological comparison; no comparative Lite Lab product data are implied.

01 / EARLIER ENTRY

Full-length SIL protein

  1. Sample
  2. Add SIL protein standard enters
  3. Process / extract
  4. Digest
  5. LC
  6. MS detection
  7. Quantification

Can accompany more of the analytical processing when added before those steps.

02 / LATER ENTRY

Heavy peptide

  1. Sample
  2. Process / extract
  3. Digest
  4. Add heavy peptide standard enters
  5. LC
  6. MS detection
  7. Quantification

Helps normalize downstream measurement; the peptide itself does not undergo protein processing or digestion.

METHOD LIMITS

Assay fit must be shown.

  • Protein form, folding, modifications, and matrix can affect comparability.
  • Losses before the spike-in point remain outside its scope.
  • Quantification also requires suitable calibration, value assignment, and validation.
SCIENTIFIC REFERENCES

Methodological context.

These publications support general principles; they do not validate Lite Lab material.

  1. 01

    Bauer Scott K, Turko IV, Phinney KW. Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry. Analytical Chemistry. 2015.

    DOI: 10.1021/acs.analchem.5b00331 ↗
  2. 02

    Lowenthal MS, Liang Y, Phinney KW, Stein SE. Quantitative bottom-up proteomics depends on digestion conditions. Analytical Chemistry. 2014.

    DOI: 10.1021/ac4027274 ↗
  3. 03

    Pratt MS, van Faassen M, Remmelts N, Bischoff R, Kema IP. An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma. Analytical and Bioanalytical Chemistry. 2021.

    DOI: 10.1007/s00216-021-03185-y ↗
NEXT STEP

Discuss your analytical workflow

Share where your current standard enters the method and what you need to evaluate.

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