The challenges of developing a sound proteomics strategy

Abstract

This paper will review the challenges of developing a proteomics strategy. A key issue is the integration of the two-dimensional (2-D) gel platform with mass spectrometry measurements. The use of both matrix-assisted laser/desorption ionization (on off-line coupling) and electrospray (on-line) ionization are complementary. While the use of one-dimensional and 2-D gels are essential to many aspects of proteomics research (sample preparation, preliminary fractionation and quantitation, storage of protein components), the emergence of shotgun sequencing based on high performance liquid chromatography and tandem mass spectrometry offers a powerful new approach. The latter has particular utility in the characterization of low level samples and complex post-translational modifications. The development of capillary columns, such as 75 to 150 micron, that can be packed in a reproducible manner has been a key step in the development of high sensitivity liquid chromatography/mass spectrometry analysis.

Sphingosine-dependent Protein Kinase-1, Directed to 14-3-3, Is Identified as the Kinase Domain of Protein Kinase Cδ

“…The conditions of MS were as follows: ion spray voltage of 1.8 kV, transfer tube temperature of 250°C for MS and MS/MS analyses, helium collision gas, and normalized collision energy of 35% for MS/MS analysis. Identification of probable sequences from MS/MS data was performed using the TurboSequest search engine (22,26,27) 4 and the NCBI Database (in house).…”

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

“…The original analytical scale size columns were supplemented with their capillary counterparts in the early 2000s [5,[12][13][14][15][16][17][18][19]. These sub-millimeter internal diameter columns facilitate direct splitless coupling with electrospray ionization mass spectrometry (ESI-MS) [20][21][22][23].…”

How Industry Is Approaching the Search for New Diagnostic Markers and Biomarkers

“…Multidimensional Chromatography-While the use of onedimensional and 2D gels is considered essential to many proteomics approaches, the emergence of shotgun sequencing based on high-performance LC and tandem MS (MS/MS) is a powerful alternative (26). Both single-dimensional highpressure LC and multidimensional LC (LC/LC) can be directly interfaced on-line with MS to allow for automated collection of large datasets (20).…”