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.
Highlights
In 1991, Desiderio and coworkers were the first to couple protein cleavage and isotope dilution mass spectrometry (PCIDMS)1 when quantifying -endorphin from human pituitary glands using a full-length (31 residue) deuterated analog [1]
To circumvent the limitations of chemical synthesis and the high cost associated with producing multiple peptides for large scale studies, Beynon and coworkers developed the QconCAT methodology in which all stable isotopelabeled (SIL) peptides are first produced as a recombinant “concatemer,” which upon digestion produces the individual SIL peptide standards [9, 10]
Theoretical Implications of Peptide Decay—PC-IDMS is based on the principle the measured concentration ratio between the native, surrogate peptide and its SIL analog accurately reflect the molar ratio between the intact protein and the SIL internal standard in the interrogated sample
Summary
Peptide Production and Decay Rates Affect the Quantitative Accuracy of Protein Cleavage Isotope Dilution Mass Spectrometry (PC-IDMS)*□S. In 1991, Desiderio and coworkers were the first to couple protein cleavage and isotope dilution mass spectrometry (PCIDMS) when quantifying -endorphin from human pituitary glands using a full-length (31 residue) deuterated analog [1] Since this seminal work, PC-IDMS has evolved and describes three distinct methods applying bottom-up methodologies (i.e. proteolysis) in conjunction with stable isotopelabeled (SIL) standards to carry out absolute quantification of proteins. We use digestion timecourse data to demonstrate that these models are sufficiently accurate and confirm that adding the SIL peptide standards concurrently with the proteolytic enzyme is the most appropriate (i.e. least biased) approach Given these findings, we propose that peptide stability be considered when selecting peptides for quantification to minimize the bias associated with peptide decay during proteolysis
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