An automated, sheathless capillary electrophoresis‐mass spectrometry platform for discovery of biomarkers in human serum

Abstract

An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serumA capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to perform routine, automated analysis of low-molecular-weight peptides in human serum. The method incorporates transient isotachophoresis for in-line preconcentration and a sheathless electrospray interface. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which peptides were added to sera from individuals at each of two different concentrations, artificially creating two groups of samples. The CE-MS data from the serum samples were divided into separate training and test sets. A pattern-recognition/feature-selection algorithm based on support vector machines was used to select the mass-to-charge (m/z) values from the training set data that distinguished the two groups of samples from each other. The added peptides were identified correctly as the distinguishing features, and pattern recognition based on these peptides was used to assign each sample in the independent test set to its respective group. A twofold difference in peptide concentration could be detected with statistical significance (pvalue , 0.0001). The accuracy of the assignment was 95%, demonstrating the utility of this technique for the discovery of patterns of biomarkers in serum.

Human body fluid proteome analysis

“…Unlike other human body fluids, removal of abundant albumin and γ-globulins from SF samples did not significantly improve 2-DE separation of SF proteins [423]. LC-MS screening of peptides in human SF indicated the presence of a peptide AGLPEKY (SAA (98)(99)(100)(101)(102)(103)(104)) derived from SAA. The peptide and several of its analogs were found capable of binding isolated human CD(4)(+) T-lymphocytes and stimulating them to produce interferon-gamma.…”

“…CE-ESI-MS is a promising tool for profiling of plasma/serum peptides [96][97][98]. The high resolving power of CE allows more than 1000 polypeptides, displayed within 45-60 min in a single analysis run.…”

Advances in Urinary Proteome Analysis and Biomarker Discovery

“…This obstacle has been resolved by improved methods of ionization and by better delivery of the separated protein from the end of a capillary to the MS instrument in a small stream of liquid by nano-ion spray. Also, improvements in the detection limits of mass spectrometers render the issue of sensitivity less important (43)(44)(45). Sequencing of potential protein biomarkers that are defined by CE-MS analysis can be achieved by directly interfacing CE with MS/MS instruments (46) or by subsequent targeted sequencing using LC-MS/MS (12,15).…”

High‐resolution proteome/peptidome analysis of peptides and low‐molecular‐weight proteins in urine

“…Improved methods of ionisation by micro-or nano-ion spray have resolved this challenge to a large extent. In addition, improvements in the detection limits of mass spectrometers enable detection in the low-or sub-femtomol range, rendering the issue of sensitivity less important [52,53,54]. Consequently, CE-MS has become a viable alternative to the commonly used proteomic technologies and has recently been successfully applied in several clinical studies [55,56,13].…”