Quantitative analysis of single amino acid variant peptides associated with pancreatic cancer in serum by an isobaric labeling quantitative method - PubMed (original) (raw)

. 2014 Dec 5;13(12):6058-66.

doi: 10.1021/pr500934u. Epub 2014 Nov 24.

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Quantitative analysis of single amino acid variant peptides associated with pancreatic cancer in serum by an isobaric labeling quantitative method

Song Nie et al. J Proteome Res. 2014.

Abstract

Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research.

Keywords: biomarker; isobaric labeling; pancreatic cancer; proteomics; serum; single amino acid variant.

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Figures

Figure 1

Figure 1

Experimental workflow for quantitative analysis of single amino acid variant peptides associated with pancreatic cancer by isobaric labeling strategy.

Figure 2

Figure 2

MS/MS spectra of the variant peptide GGGAGFISGL_TYLELDNPAGNKR (A) and canonical peptide GGGAGFISGLS_YLELDNPAGNKR (B) from Complement component C7 (CO7). The underlined and italic letter indicates the variant site. A +14 Da mass shift corresponding to the substitution of threonine (Thr) for serine (Ser) at position 389 was indicated by the series of y13, y14, y15, y16 and y17 ions, which confirms the variant site.

Figure 3

Figure 3

Variant peptide (SDNCEDTPEAGYFA_V_AVVK) from serotransferrin was verified and quantified by spiking heavy labeling peptide into serum samples. The TIC of the variant peptide (A), standard curve (B) and scatter plot of the amount of variant peptide in different disease groups (C).

Figure 4

Figure 4

Performance of variant peptide based on the results of SRM assay. the ROC curve and AUC value are presented.

Figure 5

Figure 5

Performance of biomarker combination of SAAV peptide and THBS1, AACT, the ROC curve and AUC value are shown.

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