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

What is a Stable Isotope Labelled protein ?

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”.

SIL proteins have the same physiochemical behavior as the target proteins analyte. The internal standard 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. This allows 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

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.

How to use it ?

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

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
  • Products are ready-to-use and packaged to be highly stable
  • Identical to analyte in sample processing
  • For use in complex sample matrices
  • Highly stable
  • Catalogue & custom options available
  • The addition of the SIL protein minimises variations in sample processing and reduces assay development time
DISCOVER OUR SIL PROTEINS

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