Protein identification system

Figure 4.8. MuDPIT platform. The MuDPIT or multidimensional gel protein identification system is an multidimensional liquid chromatography (LC)-based system of separation prior to tandem mass spectrometry (MS/MS). It is not necessary to purify proteins prior MuDPIT, although a reduction in protein complexity by some prior purification is helpful in obtaining interpretable spectra. Protein lysates are digested into peptides that are loaded onto a strong cation exchange (SCX) support. Peptides are sequentially eluted onto a reverse-phase column for a second separation. Eluted peptides from the reverse-phase column are electrosprayed into a tandem mass spectrometer for amino acid sequencing and identification of proteins in the sample.

Table 1. List of available high throughput protein identification systems... 

BACTERIAL PROTEIN BIOMARKER DISCOVERY A FOCUSED APPROACH TO DEVELOPING MOLECULAR-BASED IDENTIFICATION SYSTEMS... 

Monolithic silica columns with various surface derivatization, including ion exchange (Xie et al., 2005), and chiral functionalities (Chen et al., 2002 Lubda et al., 2003 Chankvetadze et al., 2004), as well as protein-immobilized monoliths (Kato et al., 2005) have been reported. The latter will be an important part of an integrated multidimensional separation/identification system. 

This chapter has presented several comprehensive 2DLC approaches combining a first-dimension IEX separation and a second-dimension RP separation for the analysis of complex protein mixtures typical in proteomics studies. Online ESI-TOF/MS detection provided sensitive detection and valuable qualitative information (MW) for proteins eluting from the MDLC system. Coordinated fraction collection and subsequent MS analysis of peptides produced by proteolysis of the fractions provided in-depth information on protein identification and a mechanism... 

J. Gao, J.D. Xu, L.E. Locascio, and C.S. Lee, Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal. Chem. 73, 2648-2655 (2001). 

W. Zhang and B. T. Chait. ProFound An Expert System for Protein Identification Using Mass Spectrometric Peptide Mapping Information. Anal. Chem., 72, no. 11 (2000) 2482-2489. 

Barth, H., Roebling, R., Fritz, M. and Aktories, K., The binary Clostridium botulinum C2 toxin as a protein delivery systems. Identification of the minimal protein region necessary for interaction of toxin, J. Biol. Chem., 277, 5074—5081, 2002. 

Fig. 3.1 Schematic of ALIS, an automated /igand identification system that uses continuous size-exclusion chromatography for the study of protein-ligand interactions.

Suzuki, T., Ito, M., Ezine, T., Shikata, M., Ando, E., Utsumi, T., Tsimasawa, S., and Nishimura, O. (2007) Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry. Proteomics , 1942-1950. 

Once peptides have been successfully eluted from multi-dimensional chromatography, they undergo CID within MS/MS instruments. Data-dependent acquisition systems associated with MS/MS allow individual peptides to be fragmented and the resultant spectra to be used to search the databases for possible identification (Wehr, 2003). This method of protein identification, via MuDPIT, is most efficient when working with genome-verified organisms (Yates, 1998). MuDPIT... 

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI)-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-... 

Davis, M. T. Lee, T. D. 1998. Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer. /. Am. Soc. Mass Spectrom., 9,194-201. 

MS, either after electrophoretic separation and proteolysis or independently of 2DE [e.g., as in Multidimensional Protein Identification Technology (MudPIT), Ref. 21, also see Sec. 3.1.3), as a stand-alone system for protein identification. 

The comprehensive molecular biology server, ExPASy (Expert Protein Analysis System), provides Proteomic tools (http //www.expasy.ch/tools) that can be accessed directly or from the ExPASy home page by selecting Identification and Characterization under Tools and Software Packages. The Protein identification and characterization tools of the Proteomics tools (Figure 11.4) provide facilities to ... 

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