Article

High sequence coverage of proteins isolated from liquid separations of breast cancer cells using capillary electrophoresis-time-of-flight MS and MALDI-TOF MS mapping.

Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Analytical Chemistry (Impact Factor: 5.7). 12/2003; 75(22):6209-17. DOI: 10.1021/ac0346454
Source: PubMed

ABSTRACT A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines. Intact proteins are isolated using multidimensional liquid-phase separations that permit the collection of individual protein fractions. Protein digests are then analyzed by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and by capillary electrophoresis-electrospray ionization (CE-ESI)-TOF MS peptide mapping. These methods can be readily interfaced to the relatively clean proteins resulting from liquid-phase fractionation of cell lysates with little sample preparation. Using combined sequence information provided by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Furthermore, an accurate intact protein MW value (within 150 ppm) can be obtained from ESI-TOF MS. The intact MW together with high coverage sequence information provides accurate identification. More notably the high sequence coverage of CE-ESI-TOF MS together with the MS/MS information provided by the ion trap/reTOF MS elucidates posttranslational modifications, sequence changes, truncations, and isoforms that may otherwise go undetected when standard MALDI-MS peptide fingerprinting is used. This capability is critical in the analysis of human cancer cells where large numbers of expressed proteins are modified, and these modifications may play an important role in the cancer process.

0 Bookmarks
 · 
54 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In recent years, several proteomic methodologies have been developed that now make it possible to identify, characterize, and comparatively quantify the relative level of expression of hundreds of proteins that are coexpressed in a given cell type or tissue, or that are found in biological fluids such as serum. These advances have resulted from the integration of diverse scientific disciplines including molecular and cellular biology, protein/peptide chemistry, bioinformatics, analytical and bioanalytical chemistry, and the use of instrumental and software tools such as multidimensional electrophoretic and chromatographic separations and mass spectrometry. In this unit, some of the common protein-profiling technologies are reviewed, along with the accompanying data-analysis tools.
    Current protocols in bioinformatics / editoral board, Andreas D. Baxevanis ... [et al.] 08/2005; Chapter 13:Unit 13.1.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.
    Journal of Proteome Research 01/2004; 3(4):801-6. · 5.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.
    Expert Review of Proteomics 10/2010; 7(5):665-78. · 3.90 Impact Factor

Full-text

View
0 Downloads
Available from