Candidate-based proteomics in the search for biomarkers of cardiovascular disease

Abstract

The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers of disease, treatment response and ageing. As the number of proteins that can be detected in plasma or serum (the primary clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease and stroke are presented as a starting point for such a 'directed proteomics' approach.

Figure 1

Data replotted from Rifai & Ridker, 2003 showing the improved discrimination of relative cardiovascular disease risk when two different markers (in this case LDL-cholesterol and C-reactive protein) are considered jointly.

Figure 2

A plot in which normal plasma concentrations for 115 proteins from Table 1 (distributed along the X-axis but unlabelled because of legibility limitations) are plotted on a log scale (pg ml⁻¹ along the Y-axis). The proteins are sorted by abundance to reveal the smooth distribution across > 10 logs of concentration. Each protein is represented by a symbol that indicates in how many of three proteomics datasets (see text) it was detected.

Figure 3

An example showing MS/MS detection of a prothrombin peptide (TATSEYQTFFNPR) in a tryptic digest of unfractionated plasma, using the SRM transition 781.4/909.7 (parent/fragment masses). Prothrombin is present in normal plasma at 100 mg ml⁻¹, and the peptide is detected at a signal-to-noise ratio (S/N, smoothed peak height/3 s background) of 85. In the figure, the arrow in panel MS1 shows the peak in the peptide MS spectrum selected as the parent, the arrow in panel MS2 shows the fragment chosen from the MS/MS spectrum (the y7 ion), and panel MRM shows the ion current detected at this parent/fragment SRM transition (with unit mass windows) over the entire course of a 3 h LC run. The MS/MS spectrum in MS2 unambiguously identifies the prothrombin peptide by sequence, providing absolute specificity better than immunoassay.