Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

Abstract

Electron transfer dissociation (ETD) is a recently introduced massspectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 (Ϸ80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single study, we were able to identify 80% of the known phosphorylation sites in >1,000 phosphorylated peptides analyzed. A hierarchical clustering of the identified phosphorylation sites allowed us to discover 15 phosphorylation motifs that have not been reported previously. Overall, ETD is an excellent method for localization of phosphorylation sites and should be an integral component of any strategy for comprehensive phosphorylation analysis.bioinformatics ͉ motifs ͉ phosphorylation ͉ signal transduction ͉ systems biology M ost cellular processes are regulated by posttranslational modifications of proteins. For some posttranslational modifications (e.g., acetylation and tyrosine phosphorylation), identifying the modified amino acid is relatively straightforward because they are quite stable in the presence of the energy required for collisioninduced dissociation (CID) experiments. For other posttranslational modifications [e.g., O-linked N-acetylglucosamine (OGlcNAc) and phosphorylated serine and threonine residues], however, localization is substantially more difficult because the peptides either lose the modification in a charge separation process (O-GlcNAc) (1-3) or by a ␤-elimination event with a neutral loss of phosphoric acid (e.g., phosphoserine into dehydroalanine). In 1998, Zubarev et al. and Pitteri et al. (6) demonstrated that peptide cations can also be reduced and converted into radicals by reaction with radical gaseous anions, in an electron transfer process. The reduced peptides show similar fragmentation patterns as observed in ECD experiments, and the process is designated electron transfer dissociation (ETD). Although ETD has been tested in pilot experiments to localize posttranslational modifications, no large-scale analysis using ETD has yet been published.In this study, we present a global proteomic profiling of phosphopeptides subjected to fragmentation using ETD in an ion trap mass spectrometer. A total of 84,000 ETD and CID tandem MS (MS/MS) spectra from 130 liquid chromatography (LC)-MS/MS runs using three different proteolytic enzymes (Lys-C, trypsin, and Glu-C) allowed us to identify 1,435 unique phosphorylation ...

Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membranes

“…A parameter file incorporating the following mass modifications was used in the search for glycated peptides: static modification for cysteine residues (carbamidomethylation, 57.02 Da), dynamic modification for methionine residues (oxidation, 16.00 Da), and dynamic modification for lysine residues (AC, 162.05 Da). Additionally, the following charge-dependent criteria that were modified from Molina et al 22 were used to filter raw search results: 5+, score >14; 4+, score >12; 3+, score >9; 2+, score >8; a percentage scored peak intensity (%SPI) >60 and Delta Rank1 − Rank2 >1 were used for all charge states. Further, each data set was searched against the same IPI database with all protein sequences reversed in order to estimate the peptide false discovery rate.…”

Analytical strategies for phosphoproteomics

“…Molina et al also compared the use of ETD and CID for fragmentation, and identified 60% more phosphopeptides when using ETD, mainly due to an average of 40% more fragment ions. However, the authors found that a combination of ETD and CID by an automatic alternating mode gave the best results as little overlap was observed from the two fragmentation techniques [85].…”

“…Furthermore, TiO 2 is extremely tolerant toward most buffers and salts used in biochemistry and cell biology laboratories [73]. This has resulted in a robust method for enrichment of phosphopeptides, which has already become a highly popular method for in large-scale phosphoproteomic studies [10,[77][78][79][80][81][82][83][84][85][86][87][88][89][90].…”

“…whereas Molina et al combined TiO 2 chromatography with ETD in a large-scale study of human embryonic kidney 293T cells, and identified 1435 phosphorylation sites of which approximately 80% were novel [85]. Molina et al also compared the use of ETD and CID for fragmentation, and identified 60% more phosphopeptides when using ETD, mainly due to an average of 40% more fragment ions.…”

“…Molina and co-workers tested the efficiency of ETD on peptides originating from trypsin or Lys-C digests and surprisingly found no major improvement through the use of Lys-C. This was thought to be due to a high level of missed cleavages in the tryptic digest caused by the presence of phosphate groups or highly acidic residues [85]. Recently, our group has presented an alternative way to increase the charge state on peptide ions.…”