Post-translational modifications (PTM) are chemical alterations to proteins that are essential to diversity of protein functions. One of the most commonly studied PTM is phosphorylation, a reversible PTM of proteins that plays a major role in the regulations of many protein functions such as cell cycle, enzyme activation/deactivation and signal transduction etc. However, quantitative analysis of phosphorylation profiling by mass spectrometry is a highly challenging task and requires enrichment for either phosphoproteins or phosphopeptides. Enrichment at the protein level will provide better protein identification with a risk of lower phosphorlyation identification. We chose to enrich at the peptide level to increase the chances of identifying phosphorylated peptides. Methods: Trypsin digested whole cell lysates of monocytes derived macrophages- were processed using TiO2 Phosphopeptide Enrichment and Clean-up Kit (Pierce, Inc.), then injected onto nano-LC-LTQ Orbitrap with ETD in configuration of HCD and ETD as two microscans complementing information from MS/MS spectra. The acquisition method was created in data-dependent mode with one precursor scan in the Orbitrap, followed by fragmentation of the 4 most abundant peaks in both ETD detected in the LTQ, and in HCD detected in the Orbitrap. Tolerances were 10ppm for the Orbitrap precursors and fragments and 0.8Da for the ETD fragments. The following dynamic modifications were applied: Phospho/+79.966Da (S, T, Y), Oxidation/+15.995Da (M), Carboxymethyl/+58.005Da (C). Data were searched using Proteome Discoverer, PEAKS Studio, OMSSA and Mascot. Preliminary data: In this study we are used human monocytes obtained from elutriation. These cells are widely accepted as a biological system to test various aspects of innate immunity responses to viral and bacterial infections. Cells were lysed using standard protocol with protease inhibitor and sodium vanadate to inhibit phosphatases. Typical lysis yields 100 μg of protein from 1×106 cells. This whole cell lysate (WCL) is subjected to overnight tryptic digest and resulting peptides are enriched using TiO2 spin columns. Preliminary results show that 100 μg of digested WCL passed through TiO2 columns yielded identification of approximately 200 phosphopeptides ranging from low to very high confidence. The number of identified phosphopeptides is dependent on the stimulation or activation of cells and the monocytes used in this study are at their resting state with reduced metabolic activity. Optimizing the yield of phosphoproteome can be multi-fold: 1. Use more initial cell lysate, 2. Use activated cells, 3. Use additional fragmentation of HCD, and 4. Improvement of data extraction. For the latter, the software available for peptide identification and localization of the phosphate group (S) will have major impact on final output. Therefore to optimize phosphoproteome profiling, we used 200 and 600 μg of initial WCL and we present here in this study the comparison of the output of 4 search algorithms on the identification of phosphopeptides. Novel aspect: To maximize identification and localization of phosphate PTM by combining multiple software packages.
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