Figure 1.
Experimental schema of the discovery and verification phases.
Left panel, serum samples collection and depletion to remove the human serum albumin (HSA); Middle panel, IMDL fractionation which separates serum peptides by their pIs (2.5∼8.5) and hydrophobicity, with an example of HPLC-MS/MS chromatography showed for each pH step elution; Right panel, the number of NSCLC patients and the number of age-matched healthy controls in the verification phase by western blotting, TMA and MRM measurement. SCX, strong cation exchanger, RP reversed-phase chromatography. AD: Adenocarcinoma; SCC: Squamous cell carcinoma; NSCLC: Non-small cell lung cancer.
Figure 2.
HCA and PCA of total serum proteomic data between samples.
(A) Hierarchical clustering analysis and (B) Principal component analysis of all the 647 proteins quantified across the 18 serum samples. Ends of red arrows in (B) represent the PCA results for each protein.
Figure 3.
A high quality serum proteome associated with NSCLC.
(A) Hierarchical clustering heatmap of 101 significantly altered proteins. Expression values are shown as a color scale with higher values represented by yellow and lower represented by blue. (B) Gene Ontology biological processes significantly enriched in 101 protein set. Gene symbols in blue or red circles (could be referred in Table S3) indicate the corresponding proteins in down- or up-regulated in NSCLC sera.
Table 1.
Serum proteins potentially relevant to NSCLC confirmed by IMDL and label free quantification in the discovery phase.
Figure 4.
TMA assisted validation for histological biomarkers.
(A) Representative IHC cases with higher expression level in NSCLC sections. One SCC and one AD sample are shown to suggest different Q scores can be obtained by the four proteins; magnification 200×. (B) Virtual result of TMA. Q scores are calculated considering both percentage of positive cells and stain intensity and shown as a color scale with higher Q represented by yellow (maximum = 27) and lower Q represented by blue (minimum = 0). The grey spot represents the only one case lost during the processing.
Figure 5.
Workflow of western blotting assisted verification for A1BG.
Western blot images across 12 ADs, 12 SCCs and 12 control sera. Coomassie stained Albumin (ALB) was shown as the loading controls. IOD values were extracted and normalized by this calibration sample, suggesting the differential distribution of A1BG serum levels between normal and lung cancer cases.
Figure 6.
The MRM measurement of serum concentrations of A1BG, LRG1 and their association with NSCLC.
(A–B) MRM Intensities of selected reference peptides of A1BG and LRG1 both showed good linear correlation with on-column abundance. The x-axis represents base-3 logarithm of ratios of spiked light and heavy isotopic peptides, with the y-axis corresponding to the observed peak area ratios in base-3 logarithmic scale. Red triangles suggest the limit of linear quantification (LOQ) of each peptide. Note that these two peptides were used to report the absolute concentration, with comparison to another two less-optimal peptides shown in Figure S4. (C–D) The chromatography peaks of the best transitions of two peptides for A1BG and LRG1. Note that the Signal-to-Noise ratios are sufficient for quantification. (E) The distribution of serum levels of A1BG and LRG1 between cancer and control groups. (F) ROC analysis suggested over-expressed A1BG and LRG1 were associated with NSCLC and their combined panel could provide the better discriminative performance than each of the protein.