Matrix-assisted laser protein identification

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

Bemdt, P., Hobohm, U., and Langen, H. (1999). Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints. Electrophoresis 20, 3521-3526. 

Dai, Y. Li, L. Roser, D. C. Long, S. R. Detection and identification of low-mass peptides and proteins from solvent suspensions of Escherichia coli by high performance liquid chromatography fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 1999,13,73-78. 

Pineda, F. J. Antoine, M. D. Demirev, P A. Feldman, A. B. Jackman, J. Longenecker, M. Lin, J. S. Microorganism identification by matrix-assisted laser/desorption ionization mass spectrometry and model-derived ribosomal protein biomarkers. Anal. Chem. 2003,75,3817-3822. 

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). 

The growing interest for the identification and characterization of polar and large compounds caused the development and the introduction of new ionization techniques, such as electrospray ionization (ESI)[4], and matrix assisted laser desorption ionization (MALDI),[5] and their more recent improvements, thus establishing new MS based approaches for studying large molecules, polymers and biopolymers, such as proteins, carbohydrates, nucleic acids. 

R. Hynek, S. Kuckova, J. Hradilova, M. Kodicek, Matrix assisted laser desorption/ionization time of flight mass spectrometry as a tool for fast identification of protein binders in color layers of paintings, Rapid. Commun. Mass Spectrom., 18, 1 5 (2004). 

A modified version of 2DE and gel image analysis, with silver staining, autoradiography, and protein identification and measurement of peptide mass, uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as a rapid and sensitive technique for identifying peptides. MALDI-TOF-MS applies well to protein detection in biological fluids.56 A second advantage of this technique is... 

Fig. 6. Protein identification using a peptide map measured with a matrix-assisted laser desorption time-of-flight mass spectrometer. All the peptide extracted from the gel is measured and the set of masses is used in the database search. The mass resolution is in the order of 10,000. Individual isotopes of a 2.5 kDa peptide are clearly resolved.

Zabet-Moghaddam, M. et al., Pyridinium-based ionic liquid matrices can improve the identification of proteins by peptide mass-fingerprint analysis with matrix-assisted laser desorption/ionization mass spectrometry. Anal. Bioanal. Chem., 384, 215, 2006. 

Kim J, Kim SH, Lee SU et al. Proteome analysis of human liver tumor tissue by two-dimensional gel eleetrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins. Electrophoresis 2002 23 4142 156. 

T. Miliotis, S. Kjellstrom, P. Onnerfjord, J. Nilsson, T. Laurell, L. E. Edholm, and G. Marko-Varga, Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry, J. Chromatogr. A, 886 (2000) 99-110. 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

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-... 

B Thiede, A Otto, U Zimny-Arndt, E-C Muller, P Jungblut. Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 17 588-599, 1996. 

KL O Connell, JT Stults. Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests. Electrophoresis 18 349-359, 1997. 

Chen, P., Nie, S., Mi, W Wang, X.-C., and Liang S.-P. (2004) De novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 18,191-198. 

Chaurand P, Luetzenkirchen F, Spengler B (1999) Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry. J Am Soc Mass Spectroml0 91-103... 

Chait, B. T. (2000). Rapidly switchable matrix-assisted laser desorption/ ionization and electrospray quadrupole-time-of-flight mass spectrometry for protein identification. J. Am. Soc. Mass Spectrom. 11, 493-504. 

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