Large-Scale Identification of Caenorhabditis elegans Proteins by Multidimensional Liquid Chromatography−Tandem Mass Spectrometry

Abstract

A proteome of a model organism, Caenorhabditis elegans, was analyzed by an integrated liquid chromatography (LC)-based protein identification system, which was constructed by microscale two-dimensional liquid chromatography (2DLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. Soluble and insoluble protein fractions were prepared from a mixed growth phase culture of the worm C. elegans, digested with trypsin, and fractionated separately on the 2DLC system. The separated peptides were directly analyzed by on-line ESI-MS/MS in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database, wormpep 66, for protein identification. The total number of proteins of the composite proteome identified in this method was 1,616, including 110 secreted/targeted proteins and 242 transmembrane proteins. The codon adaptation indices of the identified proteins suggested that the system could identify proteins of relatively low abundance, which are difficult to identify by conventional 2D-gel electrophoresis (GE) followed by an offline mass spectrometric analysis such as peptide mass fingerprinting. Among the approximately 5,400 peptides assigned in this study, many peptides with post-translational modifications, such as N-terminal acetylation and phosphorylation, were detected. This expression profile of C. elegans, containing 571 hypothetical gene products, will serve as the basic data of a major proteome set expressed in the worm.

Mycobacterium tuberculosisFunctional Network Analysis by Global Subcellular Protein Profiling

“…The unbiased nature of this technique has allowed the identification of different protein classes, including both hydrophobic and membrane proteins. It has been previously used for the sequence analysis of protein complexes and the proteomic profiling of other organisms (Takahashi et al, 1985;Link et al, 1999;Washburn et al, 2001;Kaji et al, 2003;Mawuenyega et al, 2003). Using 2DLC/MS we provide a comprehensive subcellular analysis of the protein complements isolated from the cell wall, membrane and cytosol of the Mtb H37Rv virulent strain.…”

“…Approximately 190 mg of cell wall protein, 13 mg of cell membrane protein, and 100 mg of cytosolic protein were recovered. The proteins in each of the fractions were deglycosylated with PNGase F as described by the manufacturer (New England Biolabs, Beverly, MA), treated with acetone to remove lipids, and digested with trypsin as previously described (Mawuenyega et al, 2003). The tryptic digests were dried in a speedvac and reconstituted in water twice and then in 5% acetonitrile, 0.1% formic acid solution, pH 3.0.…”

Multidimensional LC Separations in Shotgun Proteomics

“…A variety of first dimensions, not all of them LC-based, have been used, including size-exclusion chromatography (SEC), strong-cation exchange (SCX), strong-anion exchange, SDS-PAGE, and isoelectric focusing (IEF) techniques (e.g., immobilized pH gradient IEF and capillary IEF). 3,9,10,14,15,[18][19][20][21][22] Several factors are important for the first dimensionsit should have a large loading capacity, be configurable with the second dimension, and have solvent compatibility with the second mode. Several of the first-dimension methods fit these criteria, whereas others are better suited for off-line applications.…”

Capture and analysis of quantitative proteomic data

“…As the different control points in the regulation of gene expression are uncovered, from global mechanisms at the level of chromatin structure and transcriptional control, through to translational mechanisms including protein modification and turnover, the ability to quantify protein levels accurately will assume increasing importance. It is widely acknowledged that modern MS, coupled with online peptide separation methods and bioinformatics, can enable hundreds or thousands of peptides to be identified in a standard proteomics experiment [1][2][3][4][5][6] -the challenge is now to do this in a quantitative fashion, and translate these into protein levels. This will support the growing field of systems biology, where different concentrations of proteins determine various states of the sample being analysed and networks of interacting molecules are developed, ultimately to predict systems level phenomena.…”