Intact protein mass spectrometry

Trauger SA, Junker T, Siuzdak G (2003) Investigating Viral Proteins and Intact Viruses with Mass Spectrometry. 225 257-274... 

It is clear that both intact cell MALDI-TOF and PFGE have their limitations. PFGE analyses probes the chromosomal DNA of microorganisms for variations in the locations of specific restriction enzyme cleavage sites, while MALDI-TOF mass spectrometry of intact cells primarily examines abundant proteins such as ribosomal proteins35 and those associated with or near bacterial cell walls.58 In order for MALDI-TOF to detect a variation, a mutation must lead to noticeable changes in the expression of cell wall—associated... 

This chapter will address the applications of protein-based bioinformatics to analysis of microorganisms introduced intact into the instrumental system for rapid processing and analysis. Strategies that require offline extraction and fractionation of proteins will not be discussed. Although the amplification of nucleic acids is a powerful approach, especially coupled with mass spectrometry,15 it requires extraction and processing, and thus is not included. 

The introduction and eventual commercialization of matrix-assisted laser desorption/ionization (MALDI) and electrospray (ESI) allowed biomarker status to be extended to proteins in 1996.15"17 With a few exceptions, ESI has been used in conjunction with extractions and high-pressure liquid chromatography (UPLC) interfaced with mass spectrometry. MALDI, on the other hand, has been widely adapted for rapid analysis of intact organisms, supported by bioinformatics.1819... 

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. 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

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. 

Wang, H., Hanash, S. (2005). Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry. Mass Spectrom. Rev. 24, 413 126. 

Trauger SA, Junker T, Siuzdak G (2003) Investigating Viral Proteins and Intact Viruses with Mass Spectrometry. 225 257-274 Trautwein AX, see Paulsen H (2004) 235 197-219 Trautwein AX, see Winkler H (2004) 235 105-136... 

J. A. Loo, C. G. Edmonds, and R. D. Smith. Primary Sequence Information from Intact Proteins by Electrospray Ionization Tandem Mass Spectrometry. Science, 248(1990) 201-204. 

R.H.H. Investigation of intact protein complexes by mass spectrometry. Mass Spectom. Rev. 2004, 23, 368-389. 

Mass spectrometry (MS) is widely used to ascertain the purity, total mass of the protein produced, and detect any covalent modifications (Cohen and Chait, 2001). Both electrospray ionization (ESI) and MALDI may be used although for intact proteins ESI has the advantage of being accurate to 1 Da. Using the simple protocol described in Protocol 2.11, the MS of whole protein samples can be readily automated without the need for sample preparation. This method has proved successful for the... 

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