Multiplex multidimensional nanoLC-MS system for targeted proteomic analyses

Abstract

The present work describes a dual-column and dual-sprayer LC-MS system for high-throughput proteomic analyses. This system consists of two precolumns for sample desalting and two analytical columns. Each column is terminated by a nanoelectrospray emitter mounted on a robotic arm enabling their sequential positioning in front of the sampling cone of the mass spectrometer. The effluent from each emitter is recorded in separate acquisition channels without detectable crosstalk. Gradient elution to both nanoLC columns is delivered by a single HPLC system via a flow splitter. The reproducibility of retention time and peak intensity of the present multiplex system were comparable to those obtainable using a single emitter configuration. Replicate injections of complex tryptic digests (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity on both columns with RSD values of less than 0.9 and 18.6%, respectively. The application of this system is demonstrated for the monitoring of protein expression changes in U937 human monocyte cells with and without phorbol ester administration. Furthermore, we also demonstrated the use of this multiplex system in a 2-D LC configuration to increase sample loading and throughput for the analysis of biomarker samples of higher complexity. Variations in peptide abundance down to two-fold change were identified across salt fractions for spiked tryptic digests present at a level of 50 fmol in 1.5 microg of plasma samples.

Peptide Identification by Database Search of Mixture Tandem Mass Spectra

“…However, as shown in the fourth column, the main advantage of spectral library search was the reduction in the number of possible peptide candidates. We also compared the MixDB's performance with an iterative strategy where: (1) distinguishing true matches from false positives in single-peptide spectra, namely: 1) Likelihood score for one peptide match: likelihood score while considering only matched peaks from one peptide.…”

“…Comparison with current database search methods indicates that our approach has better or comparable sensitivity and precision at identifying single-peptide spectra while simultaneously being able to identify 38% more peptides from mixture spectra at significantly higher precision. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.010017, 1-11, 2011. Over the past several years there have been substantial advances in the sensitivity of protein identification approaches thanks to technological developments in chromatography (1) and tandem mass spectrometry (2,3). In shotgun proteomics, researchers can routinely identify thousands of proteins from complex biological samples in a single experiment (4 -6).…”

“…But, despite this rapid progress, there are still challenging issues that remain unsolved (7,8). One such challenge is that in any high throughput tandem MS (MS/MS) 1 experiment only a fraction of MS/MS spectra can be identified by current computational methods. Although there are many factors contributing to this low spectrum identification rate, recent studies suggest that one reason is the occurrence of co-eluting peptides.…”

Detection of co-eluted peptides using database search methods

“…The protein was finally assembled [ 1 ] by joining sequenced peptides that have overlapping amino acids, as in the "shotgun-sequencing" of DNA. Technological improvements in the area of chromatography [ 2 ] and mass spectrometry (MS) [ 3 - 6 ], in particular tandem mass spectrometry (MS 2 ), have revolutionized peptide sequencing and protein identification. In 1953, Frederick Sanger successfully sequenced the first protein, the bovine polypeptide hormone insulin.…”

Proteomics analysis of an UBE3A knockout mouse modelling Angelman syndrome

“…The sample amount needed for gel-independent methods is small if compared to 2-DE and its multiplex platform allowed the separation and identification of proteins at the same isoelectric point and molecular mass. The gel-independent platform is also benefited by its ability to mine complex data with higher throughput and good reproductivity (36). Nonetheless, the drawbacks of ICAT technique include inability to quantify proteins lacking cysteins and is limited by chromatographic sample capacity, the analysis time as well as the dynamic range of peptide detection (36,37).…”