Complex proteomes

Zuo, X., Speicher, D. W. (2000). A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal Biochem. 284(2), 266-278. 

Vollmer, M., Horth, P., Nagele, E. (2004). Optimization of two-dimensional off-line LC/MS separations to improve resolution of complex proteomic samples. Anal. Chem. 76, 5180-5185. 

A comparison of theoretical and practical peak capacity values, summarized in Table 12.2, leads to a conclusion that even the most promising 2DLC setups do not provide for the peak capacity needed to fully resolve a complex proteomic sample. As a result, the eluent entering the MS source typically contains multiple coeluting peptides. 

Activity-based protein profiling (ABPP) is a chemical proteomic strategy in which active-site-directed covalent probes are used to profile the functional states of enzymes in complex proteomes. Activity-based probes (ABPs) can distinguish active enzymes from their inactive zymogens or inhibitor-bound forms. They contain a reactive group intended to modify enzyme active sites covalently and a reporter group (typically rhodamine or biotin) that assists in detection and identification of protein targets. 

This multidimensional protein identification technology (MudPIT) specifically incorporates a strong cationic exchange (SCX) column in tandem with an RP column to achieve maximal resolution and exquisite sensitivity. MudPIT is effective for studying complex proteomes such as mammalian cellular samples. It has been applied to large-scale protein characterization with identification of up to 1484 proteins from yeast in a single experiment.12... 

The protein posttranslational modifications (PTMs) play a crucial role in modifying the end product of expression and contribute towards biological processes and diseased conditions. Important posttranslational modifications include phosphorylation, acetylation, glycosylation, ubiquitination, and nitration [Mann and Jensen, 2003], The analysis of posttranslational modifications on a proteome scale is still considered an analytical challenge [Zhou et al., 2001] because of the extremely low abundance of modified proteins among very complex proteome samples. 

Zuo X, Speicher DW. Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis. Proteomics 2002 2 58-68. 

Patricelli MP, Giang DK, Stamp LM, Burbaum JJ. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 2001 1 1067-1071. 

Leung D, Hardouin C, Boger DL, Cravatt BF. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat Biotechnol 2003 21 687-691. 

Although these probes show potent, heat-sensitive labeling profiles with purified tyrosine phosphatases, suggestive of specific active site modification, their use in complex proteomes was not reported [109]. Zhang and coworkers introduced a more specific class of tyrosine phosphatase probe [110]. This probe consists of an a-bromobenzylphosphonate moiety that acts as a tyrosine phosphate mimic and a mechanism-based inhibitor of tyrosine phosphatases. The chemistry of the probe mode of action is shown in Scheme 7. 

The probes labeled purified LacZ, a glycosidase expressed in E. coli, as weh as several structurally unrelated glycosidases in complex proteomes [114, 115]. 

By adapting these inhibitors to act as ABPP probes, Thomoson and coworkers have synthesized a series of probes that were capable of highly selective labeling of both PAD4 as well as PAD4 binding proteins in complex proteomes [127, 128]. 

Kidd D, Liu Y, Cravatt BF (2001) Profiling serine hydrolase activities in complex proteomes. Biochemistry 40 4005 -015... 

Adam GC, Burbaum J, Kozarich JW et al (2004) Mapping enzyme active sites in complex proteomes. J Am Chem Soc 126 1363-1368... 

In addition to the attempts at unraveling the modification site, Dive and coworkers also constructed two biotinylated A/BPs, 22 and 23 (Scheme 3c), and used these to study the difference in affinity- and photoaffinity MMP enrichment from a complex proteome [57]. For this, tumor extracts were spiked with hMMP-12 and hMMP-8, after which compounds 22 and 23 were applied, followed by streptavidin-coated magnetic beads for MMP pull-down. Affinity-based labeling with 23 appeared superior to photoaffinity-based labeling with 22 in terms of quantity of captured MMPs and identification of the tryptic fragments by mass spectrometry. 

In the recent literature, many examples of A/BPs containing benzophenones can be found. A first example concerns the study of HDACs. These enzymes catalyze the hydrolysis of acetylated lysine amine side chains in histones and are thus involved in the regulation of gene expression. There are approximately 20 human HDACs, which are divided into three classes (I, II, and III). Class I and II HDACs are zinc-dependent metallohydrolases that do not form a covalent bond with their substrates during their catalytic process, which is similar to MMPs. It has been found that hydroxamate 65 (SAHA, see Fig. 5) is a potent reversible inhibitor of class I and II HDACs. In 2007, Cravatt and coworkers reported the transformation of SAHA into an A/BP by installment of a benzophenone and an alkyne moiety, which resulted in SAHA-BPyne (66) [73]. They showed that the probe can be used for the covalent modification and enrichment of several class I and class II HDACs from complex proteomes in an activity-dependent manner. In addition, they identified several HDAC-associated proteins, possibly arising from the tight interaction with HDACs. Also, the probe was used to measure differences in HDAC content in human disease models. Later they reported the construction of a library of related probes and studied the differences in HDAC labeling [74], Their most... 

To address this problem, recently a new strategy for proteome analysis has emerged. This technology, named Combinatorial Proteomic, uses antibody libraries as probes to profile the expression and function of protein families in complex proteomes. The use of antibodies allows the detection of iper- and ipo-expressed proteins, even if they are at pico-quantity level, overcoming one of the proteomic limitations of difficulty in detecting low abundance proteins [46, 47],... 

Darley-Usmar, V. M. (2002). ffigh throughput two-dimensional blue-native electrophoresis a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2, 969-977. 

Chen, H., Rejtar, T., Andreev, V., Moskovets, E. and Kaiger, B.L. (2005a) Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS exclusion of redundant peptides from MS/MS analysis in replicate mns. Anal. Chem. 77, 7816-7825. 

Sieber SA, Mondala TS, Head SR, Cravatt BF. Microarray platform for proliUng enzyme activities in complex proteomes. J. Am. Chem. Soc. 2004 126 15640-15641. 

G. Choudhary, S.-L. Wu, P. Shieh, W.S. Hancock, Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trapMS-MS, J. Proteome Res., 2 (2003) 59. 

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