Influence of the Digestion Technique, Protease, and Missed Cleavage Peptides in Protein Quantitation

Abstract

Quantitative determination of absolute and relative protein amounts is an essential requirement for most current bottom-up proteomics applications, but protein quantitation estimates are affected by several sources of variability such as sample preparation, mass spectrometric acquisition, and data analysis. Among them, sample digestion has attracted much attention from the proteomics community, as protein quantitation by bottom-up proteomics relies on the efficiency and reproducibility of protein enzymatic digestion, with the presence of missed cleavages, nonspecific cleavages, or even the use of different proteases having been postulated as important sources of variation in protein quantitation. Here we evaluated both in-solution and filter-aided digestion protocols and assessed their influence in the estimation of protein abundances using five E. coli mixtures with known amounts of spiked proteins. We observed that replicates of trypsin specificity digestion protocols are highly reproducible in terms of peptide quantitation, with digestion technique and the chosen proteolytic enzyme being the major sources of variability in peptide quantitation. Finally, we also evaluated the result of including peptides with missed cleavages in protein quantitation and observed no significant differences in precision, accuracy, specificity, and sensitivity compared with the use of fully tryptic peptides.

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

“…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). For the peptide (AAVPDAVGK) selected for Na/K-ATPase quantification by BPh, the C-terminal lysine (K) is closely followed by an arginine (R), separated by a cysteine (C) residue [K.CR.…”

“…However, in a comparable study by , there was also an impact of peptide choice on OATP1B1 abundance using two of the same peptides as Terasaki et al (2014), yet little influence of peptide choice for P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), OATP1B3, and OATP2B1 was observed. The digestion strategy employed might also influence the capacity to quantify protein abundances and to generate peptides that suffer missed cleavage by trypsin (Chiva et al, 2014). Studies comparing different proteomic approaches within a laboratory found that when comparing immunoblotting to stable isotope labeling of amino acids in culture/mammals (SILAC/SILAM) techniques, there was a reasonable agreement within the same samples to the absolute quantification (AQUA) method routinely employed (Kamiie et al, 2008;Qiu et al, 2013;Prasad and Unadkat, 2014a).…”

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…Sample preparation can also contribute to this variability, with proteins either quantified directly in whole cell/tissue lysate (Weiß et al, 2015;Wi sniewski et al, 2016) or in enriched subcellular fractions Gröer et al, 2013). In addition, differences in proteolytic strategies, efficiency of protein and peptide recovery and LC-MS analysis of peptides have also been suggested to introduce bias into abundance measurements (Chiva et al, 2014;Harwood et al, 2015). Other studies suggested that different analytical methods used in the same laboratory setting can generate reasonably consistent measurements (Qiu et al, 2013;.…”

Peptide Selection for Targeted Protein Quantitation

“…For this purpose we used an already published dataset from our group consisting of a mixture of thirty commercial proteins spiked in an E. coli background. 12 Briefly, five mixes were prepared in triplicate containing different ratios of the spiked-in proteins in the E. coli background, and the samples were subjected either to an in-solution or a filter-aided digestion with trypsin prior shotgun mass spectrometry acquisition. Initially we assessed the frequency, extend and reproducibility of the most common peptide chemical modifications from the E. coli proteome background for both the in-solution and filteraided original datasets (Table 1).…”