MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides

Abstract

Background: The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented.

Methods: MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide.

Results: In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing approximately 1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000.

Conclusion: Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins.

Figure 1

Proteomic maps. (A) Theoretical distribution of the human proteins according to the SwissProt database (~25,000 genes); (B) Experimental distribution of the 1,572 proteins from the MCF-7 library (all proteins were identified with p < 0.001 and ≥ 2 spectral counts).

Figure 2

Charts that illustrate the impact of protein size on likelihood of detection. (A) Chart illustrating the distribution of observable (theoretical) tryptic peptides as a function of protein MW, for the set of 1,572 proteins; (B) Chart illustrating the ratio of the experimental percentage of identified proteins to the theoretical percentage of proteins vs. the number of amino acids in a protein. The experimental protein percentages were calculated relative to the total number of identified proteins, and the theoretical percentages were calculated relative to the total number of proteins in the SwissProt database.

Figure 3

Charts that illustrate protein abundance as a function of protein MW, for the set 1,572 proteins. (A) Chart illustrating protein abundance as a function of MW in terms of experimental protein sequence coverage; (B) Chart illustrating protein abundance as a function of MW in terms of observed/observable unique peptides; (C) Chart illustrating protein abundance as a function of MW in terms of spectral counts/observed unique peptides.

Figure 4

Extracted ion chromatograms illustrating five MRM transitions/peptide for the identification of putative cancer biomarkers in whole cellular extracts. Conditions: MCF-7 whole cellular extracts were digested with trypsin, cleaned-up with SPEC-PTSCX and SPEC-PTC18 cartridges, and analyzed by a ~4 h long LC-MS/MS gradient. The top-down order of EICs reflects the order of the five transitions shown in Table 1.