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. 2017 Jan 24:7:41191.
doi: 10.1038/srep41191.

SWATH-based proteomics identified carbonic anhydrase 2 as a potential diagnosis biomarker for nasopharyngeal carcinoma

Yanzhang Luo  1 , Tin Seak Mok  2 , Xiuxian Lin  2 , Wanling Zhang  1 , Yizhi Cui  1 , Jiahui Guo  1 , Xing Chen  1 , Tao Zhang  2 , Tong Wang  1
Affiliations

SWATH-based proteomics identified carbonic anhydrase 2 as a potential diagnosis biomarker for nasopharyngeal carcinoma

Yanzhang Luo et al. Sci Rep. .

Abstract

Nasopharyngeal carcinoma (NPC) is a serious threat to public health, and the biomarker discovery is of urgent needs. The data-independent mode (DIA) based sequential window acquisition of all theoretical fragment-ion spectra (SWATH) mass spectrometry (MS) has been proved to be precise in protein quantitation and efficient for cancer biomarker researches. In this study, we performed the first SWATH-MS analysis comparing the NPC and normal tissues. Spike-in stable isotope labeling by amino acids in cell culture (super-SILAC) MS was used as a shotgun reference. We identified and quantified 1414 proteins across all SWATH-MS analyses. We found that SWATH-MS had a unique feature to preferentially detect proteins with smaller molecular weights than either super-SILAC MS or human proteome background. With SWATH-MS, 29 significant differentially express proteins (DEPs) were identified. Among them, carbonic anhydrase 2 (CA2) was selected for further validation per novelty, MS quality and other supporting rationale. With the tissue microarray analysis, we found that CA2 had an AUC of 0.94 in differentiating NPC from normal tissue samples. In conclusion, SWATH-MS has unique features in proteome analysis, and it leads to the identification of CA2 as a potentially new diagnostic biomarker for NPC.

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Figures

Figure 1
Figure 1. SWATH and super-SILAC-based MS identifications.
(a) Venn diagram comparison of identification numbers. (b–d) Protein physical-chemical feature distributions, regarding isoelectric points (b) and charges at physiological conditions (c) and molecular weights (d). Data are presented with box-and-whisker plot, and 5% outliers are shown with red dots. A total of 1414 proteins were randomly sampled from the super-SILAC identifications and background human proteome, respectively, for the comparison with the SWATH MS identifications. *P < 0.01, as compared with any of the other groups, two-tailed Kolmogorov–Smirnov test.
Figure 2
Figure 2. Determination of differentially expressed proteins in SWATH-MS.
(a) Contour plots of ln (rowCV) versus ln (rowMean) scatter plots and the regression fitting analysis with PLGEM model. Black circles indicate the modeling points used to fit a PLGEM model. (b) Quantile-quantile (Q-Q) plot. (c) Correlation of signal-to-noise ratios (STN) and protein abundance fold changes. (d) A volcano plot showing the relationship between the PLGEM-STN and the p-value calculated by PLGEM. The DEPs are shown in red (P < 0.01). (e) DEP fold change correlation of SWATH-MS and super-SILAC MS analyses. (f) Cluster analysis using DEPs. C represents subjects with NPC, and N stands for normal subjects.
Figure 3
Figure 3. Upstream regulator analysis with IPA.
(a,b) Upstream analysis of DEPs from SWATH-MS (a) and super-SILAC MS (b). Activated (z-score ≥ 2) and inhibited (z-score ≤ −2) upstream regulators are highlighted in orange and blue, respectively. (c,d) IFNG regulated proteins in SWATH-MS (c) and super-SILAC MS (d) analyses. Up-regulated and down-regulated proteins are highlighted in red and green, respectively, and the color depth is correlated to the fold change. Orange and blue dashed lines with arrows indicate indirect activation and inhibition, respectively. Yellow and gray dashed lines with arrows depict inconsistent effects and no prediction, respectively.
Figure 4
Figure 4. SWATH-MS spectra of CA2.
(a) A shotgun spectrum of a CA2 unique peptide. The mass tolerance is shown for each detected fragment ion. (b) Retention time for the SWATH detected fragment ions. The black arrow indicates the retention time referenced by the parent ion detected in the shotgun mode. (c) The m/z distribution of SWATH detected fragment ions. The theoretical peaks of the CA2 ions were indicated by the vertical red lines, while the experimentally detected CA2 y-ions were specified.
Figure 5
Figure 5. Diagnostic power verification of CA2.
(a) Immunoblotting analysis on the CA2 expression in NPC and normal tissues. Data are shown as mean ± s.e.m. (b) Representative immunohistochemical images of CA2 staining in a tissue array of nasopharyngeal tissue from normal and NPC donors. (c) Statistical analysis of CA2 expressions. The Histologic Scores of each group are shown in a box-and-whisker plot. *P = 5.28 × 10−6, donor of normal n = 13, donor of NPC n = 52, two-tailed Kolmogorov–Smirnov test. (d) Bootstrap comparison of the CA2 expression. The star indicates the mean Histologic Score of each group, while the horizontal line depicts 95% confidence interval of the mean value acquired from 10000 resampled Histologic Scores. (e) The ROC curve generated from the Histologic Scores of all donors.
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