Absolute Quantification of Proteins by Mass Spectrometry

Abstract

Mass spectrometry (MS) provides unambiguous molecular specificity relative to all other analytical and immunological techniques and, consequently, has great potential to provide unambiguous estimates of absolute protein quantities within complex biological systems. Here, we provide a critical review and discussion of the predominant MS ‐based technology used for absolute protein quantification, referred to as protein cleavage‐isotope dilution mass spectrometry ( PC‐IDMS ). This technique requires proteolytic conversion of the analyte protein into its quantitative surrogate peptide, which is subsequently quantified using a stable isotope‐labeled (SIL) peptide analog as an internal standard (IS). All phases of the PC‐IDMS workflow, from the SIL peptide production platforms to data analysis, are described and characterized in detail. An emphasis is placed on the practical quantitative aspects for each step of the workflow and their implication on quantitative accuracy.

Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised

“…The conventional hierarchy of standards (CS and IS) during bottom-up protein analysis has been established to be fulllength SIL proteins (SIL proteins, best), enzymatically cleavable SIL surrogates (cSILs), and tryptic SIL peptides (tSILs, worst). 3 Despite early reports that IC with tSILs routinely provide "absolute" (i.e., accurate) protein concentration assignments, 4,5 it is now generally accepted that accuracy of IC with tSILs requires complete digestion of the target protein into the signature peptide (or perhaps more aptly, "surrogate peptide" in applications of tryptic peptide calibrators). 6 Enzymatically cleavable surrogates, such as concatemers, 7 cleavable peptides, 8 or partial proteins, 9 have consequently been utilized during IC to facilitate absolute accuracy by normalizing variance in enzymatic digestion, particularly by emulating the native protein sequence in the flanking regions on either termini of the signature peptide(s).…”

“…The conventional hierarchy of standards (CS and IS) during bottom-up protein analysis has been established to be full-length SIL proteins (SIL proteins, best), enzymatically cleavable SIL surrogates (cSILs), and tryptic SIL peptides (tSILs, worst) . Despite early reports that IC with tSILs routinely provide “absolute” (i.e., accurate) protein concentration assignments, , it is now generally accepted that accuracy of IC with tSILs requires complete digestion of the target protein into the signature peptide (or perhaps more aptly, “surrogate peptide” in applications of tryptic peptide calibrators) .…”