Fast proteomic protocol for biomarker fingerprinting in cancerous cells

Abstract

The advance of novel technologies that will enable the detection of large sets of biomarker proteins, to greatly improve the sensitivity and specificity of an assay, represents a major objective in biomedical research. To demonstrate the power of mass spectrometry (MS) detection for large-scale biomarker screening in cancer research, a simple, one-step approach for fast biomarker fingerprinting in complex cellular extracts is described. MCF-7 breast cancer cells were used as a model system. Fast proteomic profiling of whole cellular extracts was achieved on a linear trap quadrupole (LTQ) mass spectrometer by one of the following techniques: (a) data-dependent liquid chromatography (LC)-MS/MS of un-labeled cell extracts, (b) data-dependent LC-MS/MS with pulsed Q dissociation (PQD) detection of iTRAQ labeled samples, and (c) multiple reaction monitoring (MRM)-MS of low abundant proteins that could not be detected with data-dependent MS/MS. The data-dependent LC-MS/MS analysis of MCF-7 cells enabled the identification of 796 proteins (p<0.001) and the simultaneous detection of 156 previously reported putative cancer biomarkers. PQD detection of iTRAQ labeled cells resulted in the detection of 389 proteins and 64 putative biomarkers. MRM-MS analysis enabled the successful monitoring of a panel of low-abundance proteins in one single experiment, highlighting the utility of this technique for targeted analysis in cancer investigations. These results demonstrate that MS-based technologies relying on a one-step separation protocol have the potential to revolutionize biomarker research and screening applications by enabling fast, sensitive and reliable detection of large panels of putative biomarkers. To further stimulate the exploration of proteins that have been previously reported in the literature to be differentially expressed in a variety of cancers, an extensive list of approximately 1100 candidate biomarkers has been compiled and included in the manuscript.

Figure 1

2D-view data dependent LC-MS/MS analysis of an MCF-7 cell extract (generated with the Xcalibur 2.2 software from Thermo Electron Corp). Conditions: MCF-7 cells were cultured in E2/DMEM; Sample injection volumes were 8 μL, and the protein concentration in the sample subjected to LC-MS/MS analysis was ~1-2 μg/μL; The LC separation column was a 100 μm i.d. × 12 cm fused silica capillary packed with 5 μm Zorbax SB-C18 particles; The composition of mobile phase A was H₂O:CH₃CN (95:5 v/v) and of mobile phase B was H₂O:CH₃CN (20:80 v/v), each containing 0.01 % CF₃COOH; The separation gradient, running from 0 to 100 % B, was 3 h long; The volumetric flow rate through the LC separation column was ~170-180 nL/min.

Figure 2

Tandem mass spectrum of a representative peptide belonging to putative biomarker protein 4F2 cell-surface antigen heavy chain (CD98 antigen). Conditions are provided in the experimental section; Specific LC-MS/MS conditions are provided in Figure 1.

Figure 3

Reproducibility of protein identifications in six replicate LC-MS/MS analyses of MCF-7 cell extracts (DMEM/E2 cultures). (A) All proteins with p<0.001 that were matched by peptides that passed the Xcorr vs. charge state filter (1.9, 2.2, 3.8) were considered in the analysis. Total unique proteins identified in six runs was 796; (B) Only proteins with p<0.001 that were matched by at least two unique peptides that passed the Xcorr vs. charge state filter (1.9, 2.2, 3.8) were considered in the analysis. Total unique proteins identified in six runs was 375. Other conditions were the same as provided in Figure 1.

Figure 4

Categorization of identified proteins in MCF-7 cell extracts (DMEM/E2 cultures) by data dependent LC-MS/MS analysis. (A) Biological process; (B) Cellular location. Conditions were the same as provided in Figure 1. The pie charts were created with MS multiconsensus results generated from six LC-MS/MS runs.

Figure 5

MRM-MS based detection of a putative biomarker peptide belonging to PCNA (ATPLSSTVTLSMSADVPLVVEYK) that is not identifiable in the MCF-7 cell extract by data dependent analysis. Conditions: Whole MCF-7 cellular tryptic digests were analyzed by a 3 h long LC-MS/MS gradient; Specific LC-MS/MS conditions are provided in Figure 1; MRM/CID conditions are provided in the experimental section. (A) EICs that reflect five characteristic transitions at the elution time of the peptide; (B) Tandem mass spectrum of the peptide (as generated by SCX-LC-MS/MS).