[Show abstract][Hide abstract] ABSTRACT: The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.
[Show abstract][Hide abstract] ABSTRACT: Nanoliter flow rate is optimum for separation in capillary column liquid chromatography coupled with tandem mass spectrometry (muHPLC-MS/MS). In order to develop a high-performance automated proteome analysis system allowing direct injection sample containing detergents, the influence of void volume varied from 0 to 5 microL on the separation performance and proteomic coverage of sample injection system using strong cation-exchange (SCX) trap column was investigated, it was found the void volume hardly affects the separation performance by using SCX trap column. Thus, a fully automated sample injection system using SCX trap column and ten-port switching valve was established for efficient shotgun proteome analysis. In this system, a nanoflow switching valve and a microtee were used to connect the SCX trap and analytical columns, and the uncleaned samples of proteolytic digests containing contaminants could be directly injected with minor influence on the separation performance, which was demonstrated to be a useful strategy in proteome analysis.
Journal of Chromatography A 12/2007; 1171(1-2):56-62. DOI:10.1016/j.chroma.2007.09.048 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In proteomic analysis, MS/MS spectra acquired by mass spectrometer are assigned to peptides by database searching algorithms such as SEQUEST. The assignations of peptides to MS/MS spectra by SEQUEST searching algorithm are defined by several scores including Xcorr, Delta Cn, Sp, Rsp, matched ion count and so on. Filtering criterion using several above scores is used to isolate correct identifications from random assignments. However, the filtering criterion was not favorably optimized up to now.
In this study, we implemented a machine learning approach known as predictive genetic algorithm (GA) for the optimization of filtering criteria to maximize the number of identified peptides at fixed false-discovery rate (FDR) for SEQUEST database searching. As the FDR was directly determined by decoy database search scheme, the GA based optimization approach did not require any pre-knowledge on the characteristics of the data set, which represented significant advantages over statistical approaches such as PeptideProphet. Compared with PeptideProphet, the GA based approach can achieve similar performance in distinguishing true from false assignment with only 1/10 of the processing time. Moreover, the GA based approach can be easily extended to process other database search results as it did not rely on any assumption on the data.
Our results indicated that filtering criteria should be optimized individually for different samples. The new developed software using GA provides a convenient and fast way to create tailored optimal criteria for different proteome samples to improve proteome coverage.
[Show abstract][Hide abstract] ABSTRACT: An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.
[Show abstract][Hide abstract] ABSTRACT: Fe3+-immobilized mesoporous molecular sieves MCM-41 with particle size of ca. 600 nm and pore size of ca. 3 nm is synthesized and applied to selectively trap and separate phosphopeptides from tryptic digest of proteins. For the capture of phosphopeptides, typically 10 microL of tryptic digest solution was first diluted to 1 mL by solution of ACN/0.1% TFA (50:50, v/v) and incubated with 10 microL of 0.1% acetic acid dispersed Fe3+-immobilized MCM-41 for 1 h under vibration. Fe3+-immobilized MCM-41 with trapped phosphopeptides was separated by centrifugation. The deposition was first washed with a volume of 300 microL of solution containing 100 mM NaCl in ACN/0.1% TFA (50:50, v/v) and followed by a volume of 300 microL of solution of 0.1% acetic acid to remove nonspecifically bound peptides. The nanoparticles with trapped phosphopeptides are mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and deposited onto the target for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that phosphopeptides from tryptic digest of alpha-casein and beta-casein are effectively and specifically trapped on Fe3+-immobilized MCM-41 with few peptides nonspecifically adsorbed. After the extraction by Fe3+-immobilized MCM-41, the suppression to the detection of phosphopeptides caused by abundant nonphosphopeptides from tryptic digest is effectively eliminated, and the detection of phosphopeptides by MALDI is greatly enhanced with the value of signal-to-noise (S/N) increased by more than an order of magnitude. It is demonstrated that the mechanism of the adsorption of phosphopeptides on Fe3+-immobilized MCM-41 is based on the interaction between the Fe3+ and the phosphate group. Finally, Fe3+-immobilized MCM-41 is applied to extract phosphopeptides from tryptic digest of the lysate of mouse liver for phosphoproteome analysis by nano-LC-MS/MS.
Journal of Proteome Research 12/2006; 5(11):3114-24. DOI:10.1021/pr0600125 · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, a gel free chemiproteomic method based on chromatography was developed and applied for the biological fingerprinting analysis of complex biological system. p-Aminobenzamidine (ABA), an inhibitor of trypsin-like serine proteases, was immobilized for characterizing their interacting proteins in human plasma. By the proteomic analysis method, 214 proteins were identified with obvious affinity to the immobilized ABA. By searching the sequences of above proteins with consensus patterns of the two active sites, seven proteins belong to trypsin-like serine protease group were found. Based on the Gene Ontology annotation, the identified trypsin-like serine proteases have the function of catalytic activity and calcium ion binding, and are mainly involved in the biological process of blood coagulation. Eight more other proteins related to calcium ion binding and blood coagulation were found. Nearly all of these proteins cannot be identified by directly analyzing the plasma sample demonstrating the chemiproteomics a useful approach to characterize interacting proteins in the low abundance range.
Journal of Chromatography A 12/2006; 1134(1-2):134-42. DOI:10.1016/j.chroma.2006.08.097 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.
Journal of Proteome Research 10/2006; 5(9):2431-7. DOI:10.1021/pr060162f · 5.00 Impact Factor