Prediction of Missed Proteolytic Cleavages for the Selection of Surrogate Peptides for Quantitative Proteomics

Abstract

Quantitative proteomics experiments are usually performed using proteolytic peptides as surrogates for their parent proteins, inferring protein amounts from peptide-level quantitation. This process is frequently dependent on complete digestion of the parent protein to its limit peptides so that their signal is truly representative. Unfortunately, proteolysis is often incomplete, and missed cleavage peptides are frequently produced that are unlikely to be optimal surrogates for quantitation, particularly for label-mediated approaches seeking to derive absolute values. We have generated a predictive computational tool that is able to predict which candidate proteolytic peptide bonds are likely to be missed by the standard enzyme trypsin. Our cross-validated prediction tool uses support vector machines and achieves high accuracy in excess of 0.94 precision (PPV), with attendant high sensitivity of 0.79, across multiple proteomes. We believe this is a useful tool for selecting candidate quantotypic peptides, seeking to minimize likely loss owing to missed cleavage, which will be a boon for quantitative proteomic pipelines as well as other areas of proteomics. Our results are discussed in the context of recent results examining the kinetics of missed cleavages in proteomic digestion protocols, and show agreement with observed experimental trends. The software has been made available at http://king.smith.man.ac.uk/mcpred.

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells

“…The peptide (AAVPDAVGK) selected by BPh to quantify Na/K-ATPase (ATP1A1) is not unique, as the sequence is also found in human Na/K-ATPase a2 subunit (ATP1A2) (Shull and Lingrel., 1987). Peptides possessing a lower potential for missed cleavage events are also advantageous (Lawless and Hubbard, 2012). This is pertinent given that in relative quantification analysis, up to 46% of peptides generated miscleaved events after in-solution trypsin digestion in E. coli (Chiva et al, 2014).…”

“…The proteotypic standard peptides analyzed by each laboratory for quantification of Na/K-ATPase, P-gp, and BCRP were selected using criteria published by Tohoku University and UoM (Kamiie et al, 2008;Russell et al, 2013). For Na/K ATPase (ATP1A1), the peptide selection by BPh (AAVPDAVGK) was not favorable for selection by UoM because of the potential for peptide miscleavage, as flagged the CONSeQuence program developed at the UoM (Lawless and Hubbard, 2012). Concerns were also raised regarding the peptides' uniqueness for quantification purposes, as AAVPDAVGK also occurs in human Na/K-ATPase a2 subunit (Shull and Lingrel, 1987), a protein expressed at low levels in human liver tissue (http://www.proteinatlas.…”

“…is not detected in other populations, including Caucasians (n = 100), Asian Americans (n = 30), Mexican Americans (n = 10), Pacific Islanders (n = 7), and Japanese (n = 145) (Kroetz et al, 2003;Ozawa et al, 2004). The standard peptide selected for P-gp quantification by BPh was flagged as having the potential for a trypsin missed cleavage event by ConSEQuence (Lawless and Hubbard, 2012). In the event of missed cleavage, the selected peptide would be elongated, changing its mass and resulting in the first mass filter (Q1) of a triple-quadrupole MS rejecting its selection for downstream collision fragmentation (Q2) and product ion monitoring (Q3).…”

“…This event was judged tolerable at UoM, given that the rate of glutamine deamidation is considerably lower than that for asparagine (N), with a reaction half-life of 660 days (Li et al, 2010b). The selection criteria employed by UoM would have rejected the BPh BCRP peptide standard owing to the anticipated difficulty of efficient trypsin cleavage at dibasic and tribasic tryptic sites, i.e., sequential lysine or arginine residues or lysine and arginine side-by-side at the N-terminal region of the BPh peptide (Lawless and Hubbard, 2012).…”

Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb, a Key Enzyme in Amylopectin Biosynthesis

“…A missed Glu-C cleavage was consistently observed around the Ser 649 site, resulting in a peptide with the sequence WIDFPRGPQR-LPSGK, which was identified with 99% confidence in searches against the Z. mays protein library. Low digestion efficiency at the third Asp of the Ser 649 peptide may be due to the Pro located two residues C-terminal to it, because Pro at this position has been shown to inhibit protease activity (45). A Ser residue is the only potential phosphorylation site within this sequence and MS/MS data acquired from phospho-WIDFPRGPQRLPSGK featured product ions consistent with serine phosphorylation (Fig.…”