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Benefits of Stable Isotope Labelled Proteins
as Internal Standards in Quantitative LC-MS workflows

A stable isotope labelled (SIL) form of an analyte protein is widely regarded as the optimal internal standard (IS) for absolute quantification of proteins using LC-MS; SIL proteins are often cited as being the “gold standard IS”.

The benefit of using a SIL protein as an internal standard is that shares the same physiochemical behaviour as the target protein analyte. This means that the IS can be introduced at the start of the sample preparation workflow and will be processed along with the analyte protein throughout the entire analytical procedure (see schematic above). The addition of SIL-proteins at the start of the sample extraction can control for variations due to immunoaffinity or other isolation techniques, enzymatic digestion, other pre-analytical treatments as well as the LC and mass spectrometric ionization.

EMA guidelines recommend the use of a SIL-protein IS whenever possible for LC-MS/MS methods. Beyond this, there is a growth in scientific literature that strongly supports the use of full length, recombinant SIL proteins as internal standards and selected publications discussing the benefits of using SIL proteins in quantitative LC-MS workflows are detailed above. Significantly, these citations illustrate that the addition of a SIL protein as an IS at the start of the sample preparation increases accuracy and precision of the quantitative LC-MS assay when compared to other IS approaches e.g. SIL peptides or SIL extended peptides.

Our stable isotope labelled (and unlabelled)  full length
recombinant protein products are especially suitable for:

  • Biomarkers evaluation and validation > Labelled proteins provide the most reliable, reproducible and accurate quantification data for quantitative LC-MS work
    > Products are ready-to-use and packaged to be highly stable.
    > To use requires a simple modification to the workflow by spiking a controlled concentration of the SIL protein minimising sample processing variation and reduces assay development time
  • Development of Certified Reference Materials
  • Pharmacokinetics studies of biotherapeutics > Promise Proteomics has an extensive experience in producing labelled therapeutic proteins and antibodies for use in PK/PD studies using LC-MS.
    > Promise has participated in collaborative R&D projects with pharmaceutical companies based on our high-level expertise in monoclonal antibody quantitation.
  • Quantification of therapeutic monoclonal antibodies in clinical research studies

Key Benefits of Promise Proteomics
stable isotope labelled proteins

  • High protein purity & isotopic incorporation
  • Flexibility in peptide selection
  • Same sequence as native protein
  • Equivalent digestion kinetics to target
  • Identical to analyte in sample processing
  • For use in complex sample matrices
  • Highly stable
  • Catalogue & custom options available

Publications Citing the Benefits of using Full Length Stable Isotope
Labelled Recombinant Proteins as Internal Standards in Quantitative LC-MS

View publications
  • Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LCMS – MS. Morse Faria and Matthew S. Halquist
  • Matrix effects and application of matrix effect factor – Bioanalysis. 2017, Nov. Vol 9, No23. Editorial
  • Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised – Anal Chem. 2017 Jul 18;89(14):7406-7415.
  • Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators – J Proteome Res. 2013 Dec 6;12(12):5760-74
  • Targeted absolute quantitative proteomics with SILAC internal standards and unlabelled full-length protein calibrators (TAQSI) – Rapid Commun Mass Spectrom. 2016 Mar 15;30(5):553-61
  • Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation – Anal Chem. 2016 Jan 5;88(1):746-53. doi: 10.1021/acs.analchem.5b03004. Epub 2015 Dec 14.
  • Effects of calibration approaches on the accuracy for LC-MS targeted quantification of therapeutic protein – Anal Chem. 2014 Apr 1;86(7):3575-84. doi: 10.1021/ac5001477
  • High-sensitivity LC-MS/MS quantification of peptides and proteins in complex biological samples: the impact of enzymatic digestion and internal standard selection on method performance – Anal Chem. 2013 Oct 15;85(20):9528-35. doi: 10.1021/ac4015116
  • Quantifying protein measurands by peptide measurements: where do errors arise? – J Proteome Res. 2015 Feb 6;14(2):928-42. doi: 10.1021/pr5011179. Epub 2015 Jan 6.
  • Quantitative bottom-up proteomics depends on digestion conditions – Anal Chem. 2014 Jan 7;86(1):551-8. doi: 10.1021/ac4027274. Epub 2013 Dec 9. – Methods Mol Biol. 2011;753:93-115. doi: 10.1007/978-1-61779-148-2_7.
  • Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry – J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Apr 15;893-894:1-14
  • Metrological traceability in mass spectrometry-based targeted protein quantitation: a proof-of-principle study for serum apolipoproteins – A-I and B100, J Proteomics. 2014 Sep 23; 109:143-61. doi: 10.1016/j.jprot.2014.06.015
  • Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry – Anal Chem. 2015 Apr 21;87(8):4429-35
  • Quantification of urinary albumin by using protein cleavage and LC-MS/MS – Clin Chem. 2009 Jun;55(6):1100-7
  • Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using their fully 15N-labeled analogues as internal standards – J Proteome Res. 2011 Aug 5;10(8):3802-13
  • Stable isotopically labelled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? – Rapid Commun Mass Spectrom. 2005;19(3):401-7.
  • Multiplexed LC-MS/MS assay for urine albumin – J Proteome Res. 2014 Sep 5;13(9):3930-9.
  • Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma – Mol Cell Proteomics. 2012 Jun;11(6):M111.013854.
  • General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies – Anal Chem. 2012 Feb 7;84(3):1267-73