Intact protein

Lim, W.A. Reading between the lines SH3 recognition of an intact protein. Structure 4 657-659, 1996. 

The digestion of the protein, after heme removal, using Glu-C endoproteinase was also carried out. This enzyme cleaves the polypeptide backbone on the carboxyl terminus of a glutamic acid residue and in this case yielded twelve chromatographic responses. Despite two of these arising from unresolved components, molecular weight information was obtained from 15 polypeptides, one of which was the intact protein, covering the complete sequence, as shown in Table 5.10. 

Recently, the related phenomenon of RNA interference (RNAi) has attracted much attention [5]. RNAi occurs when a short (generally 21 nucleotides in length) double-stranded RNA (dsRNA) catalyticaUy represses the translation of a fully complementary mRNA sequence. The process appears to proceed via a complex formed between the antisense RNA strand and a protein with RNase activity [6]. Upon binding to the target mRNA sequence, the ribonucleoprotein complex initiates cleavage of the mRNA transcript thus preventing translation of intact protein. After dissociation from the truncated mRNAs, the ribonucleoprotein complex is free to act on other intact mRNAs. Such small interfering RNAs (siRNAs) have... 

Romani N, Koide S, Crowley M. Witmer-Pack M. Livingstone AM, Fathman CG. Inaba K. Steinman RM Presentation of exogenous protein antigens by dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal Langerhans cells. J Exp Med 1989 169 1169-1178. 18... 

For these reasons we have developed a different approach that measures differential expression of intact proteins.21 In this approach the proteins are extracted from the cell, separated on an HPLC column, ionized via electrospray, and automatically deconvoluted into their respective uncharged nominal masses. By this methodology it is then possible to obtain accurate, intact protein profiles of the individual strains of bacteria. Because the masses of the detected proteins are accurate to +2 Da from run to run, it is possible to subtract protein profiles from known strains to quickly identify differences in protein expression among newly mutated strains. 

Williams, T. L. Cahahan, J. H. Monday, S. R. Feng, P. C. Musser, S. M. Relative quantitation of intact proteins of bacterial ceh extracts using coextracted proteins as internal standards. Anal. Chem. 2004, 76,1002-1007. 

Demirev, P. A. Ramirez, J. Fenselau, C. Tandem mass spectrometry of intact proteins for characterization of biomarkers from Bacillus cereus T spores. Anal. Chem. 2001, 73, 5725-5731. 

Mortz, E. O Connor, P. B. Roepstorff, P. Kelleher,N. L. Wood,T. D. McLafferty, F. W. Mann, M. Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence data bases. Proc Nat. Acad. Sci. USA 1996, 93, 8264-8267. 

Cargile, B. J. McLuckey, S. A. Stephenson, J. L. Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions. Anal. Chem. 2001, 73,1277-1285. 

It was in this context that the first true comprehensive online LC x LC separation was reported (Bushey and Jorgenson, 1990). Mixtures of intact proteins were analyzed using cation-exchange chromatography (CEX) as the first dimension and size exclusion chromatography (SEC) as the second. This research demonstrated that the practical difficulties of coupling two dissimilar LC modes for a comprehensive 2D separation are relatively easy to overcome when instrumentation is properly configured. 

This instrument was used to analyze mixtures of intact proteins, including protein standards and a cell lysate of the bacterium Escherichia coli. A UV... 

The SEC x RPLC approach was also used to separate intact proteins rather than peptides, in a collaborative effort with Arthur Moseley at Glaxo Wellcome, Inc. (Opiteck et al. 1998b). To optimize the size exclusion separation for proteins, six columns with 125 A pores were exchanged for a series of eight columns with 250 A pores or a combination of six columns with 250 A pores and six columns with 450 A... 

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

Liu, H. J., Berger, S. J., Chakrahorty, A. B., Plumh, R. S., Cohen, S. A. (2002). Multidimensional chromatography coupled to electrospray ionization time-of-fhght mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J. Chromatogr. B 782(1-2), 267-289. 

The enormous dynamic range of proteins in the sample represents an additional difficulty in proteome analysis. The best example is semm with a protein abundance ranging over eleven orders of magnitude (Anderson and Anderson, 2002). To detect the low abundant species, one has to load a sufficient amount of digest on a column to meet the limit of detection (LOD) of the MS instrument. Some reports published used up to 2.5 L of plasma with an extensive fractionation of intact proteins prior to LC-MS analysis on the peptide level (Rose et al., 2004). 

The ESI-MS of an intact protein yields a series of ions with mfc values corresponding to sequentially charged species (Fenn et al., 1989). Algorithms and software for the deconvolution of these peaks into a single neutral mass have been available for many... 

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

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