Natural Flanking Sequences for Peptides Included in a Quantification Concatamer Internal Standard

Abstract

Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven (15)N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide. Individual QconCATs were mixed with recombinant human clusterin at a 1:1 molar ratio and digested, and the actual ratios for each combination of peptide/flanking sequence were measured with a multiple reaction monitoring assay. Data analysis suggested that natural flanking sequences shorter than +6 residues can cause a quantitative error because the random appearance of other amino acid residues in close proximity to trypsin cleavage sites has unpredictable consequences for the digestion rates of QconCATs.

Determining Allele-Specific Protein Expression (ASPE) Using a QconCAT-Based Proteomics Method: a Novel Approach to Identify Cis-acting Genetic Variants

“…Thus, digestion efficiency of the concatenated surrogate peptides in a conventional QconCAT protein can differ significantly from that of the peptides in native proteins, which imposes significant challenges on the accuracy and repeatability of QconCAT-based quantitative proteomics. Cheung et al assessed the effect of natural amino acid flanking sequence on trypsin digestion efficiency of QconCAT proteins and concluded that including six or more amino acid flanking residues is necessary for reliable quantification 30 . In the preset study, we included at least 15 native flanking amino acids to ensure accurate measurement of any departure of ASPE ratios from one.…”

A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide‐concatenated standards

“…Although we have not evaluated digestion efficiency directly, these results indirectly support similar digestion efficiencies between the target and standard proteins and between the tertiary PCSs and HSA, regardless of background proteins, and thereby the reliability of the quantification presented here. In the meanwhile, as amino acid residues surrounding the cleavage sites of more than three have been reported to have potential effect on missed cleavage , longer flanking sequences (e.g., six residues) might be required for more identical digestion efficiencies of the target and standard proteins. The stability and degradation of peptides can also provide a bias for absolute measurement.…”

Assessment of Extracellular Vesicles Purity Using Proteomic Standards

“…14,15,18,19 The amino acid sequences of EXO1 and EXO2 QconCATs designed for quantification of EVs proteins are shown in Figure S1 (Supporting Information). The synthetic genes encoding these sequences were synthesized and incorporated into the pET21a expression vector, with codon optimization for E. coli (Biomatik, Cambridge, Ontario).…”