title: Peptide production and decay rates affect the quantitative accuracy of protein cleavage isotope dilution mass spectrometry (PC-IDMS).
abstract: No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the protein quantities measured in protein cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.
authors.affiliation: W M Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Shuford
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
mesh_terms: D007201:Indicator Dilution Techniques; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010452:Peptide Biosynthesis; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D032107:Populus / Q000201:enzymology* / Q000378:metabolism; D059748:Proteolysis; D052584:Xylem / Q000201:enzymology*
chemical_list: D010455:Peptides