Identification of Proteins by Mass Spectrometry

The number of different protein species in plasma is in the hundreds of thousands with some estimates as high as 500 000 all of these are ultimately derived from the genetic blueprint carried in the DNA of all cells in the individual organism, but in humans the number of identifiable genes is only 30000. One important 

A second dimension of separation can then he achieved in a direction perpendicular to that used for the pi separation by connecting the original strip to a plate that is free of ampholytes in the polyacrylamide gel layer. Application of an appropriate field can now achieve a second separation on the basis of molecular mass provided that certain conditions are satisfied. The electrophoretic mobility, and thus the speed of motion in this second dimension, depends not only on the nett charge but also on the hydrodynamic radius (basically determined by the overall size and 3D shape) of the protein molecule (Section 3.7). 

Since its original introduction (Laemmli 1970) this combination of polyacrylamide gel electrophoresis (PAGE) with denaturation by SDS has become well established but further improvements to the SDS-PAGE method are still being developed (e.g., Schagger 1987). Note that getting rid of the SDS detergent is essential before LC—MS/MS analysis is attempted, since the dodecylsulfate anions form ion pairs with the protonated peptides and severely reduce the electrospray response of the latter. 

The preceding discussion may seem like a long preamble concerning analyte identification before discussing the quantitation methods that are the true subject of interest here but, in the case of quantitative proteomics, identification can be a major issue (Nesvizh-skii 2005). 

---

Without any doubt, mass spectrometry is now the most efficient way to identify proteins [75-78], The method is based on comparison of the data obtained from the mass spectrometry with those predicted for all the proteins contained in a database. The efficiency of the method results from the development of mass spectrometry into a rapid and sensitive method to analyse peptides and proteins and also from the availability of larger and larger databases. In October 2006, these databases contained more than 2400000 non-redundant sequences. Furthermore, the data obtained from genomic sequences after translation in the six lecture frames also can be used. The databases based on expressed sequence tags (ESTs) are another usable source for search. They are composed of sequences based on cDNA fast sequencing. They are limited to short lengths, about 300 bases, and contain many errors but they correspond to coding sequences. Despite their defects, they are very useful for identification of proteins by mass spectrometry [79]. 

Current proteomic methods of protein separation by 2D gel and/or high-performance liquid chromatography (HPLC), as well as the identification of proteins by mass spectrometry (MS) in conjunction with protein databanks and several software programs are satisfactory. These methods must be improved in the future to be more efficient. Some improvements that are expected in the near future must include the following strategies as discussed by others as well ... 

Knowledge about the subcellular localization of a protein may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed subcellular proteomics. ... 

The microreactors require mixing and dispersion of various reactants. In a lab-on-a-chip system, it is often required to mix reactants. One example application is fragmentation of proteins by mixing them with enzymes. This procedure is used for the identification of proteins by mass spectrometry. The role played by hydrodynamic instabilities and turbulence on mixing between two streams is limited or nonexistent due to the low Reynolds number in microsystem. The detailed understanding of diffusion and dispersion is necessary to investigate the mixing issues in microsystems. These issues have been discussed in this chapter. 

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. 

Figeys, D. Aebersold, R. High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry inital comparison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer. Electrophoresis 1997,18, 360-368. 

Strategy for Identification of Sites of In vivo Post-Translational Modifications of HMGAl Proteins by Mass Spectrometry... 

Sickmann, A., Mreyen, M. and Meyer, H.E. (2002) Identification of modified proteins by mass spectrometry. lUBMB Life,... 

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. 

D. Figeys, A. Ducret and R. Aebersold, Identification of proteins by capillary electrophoresis-tandem mass spectrometry , Evaluation of an on-line solid-phase extraction device , J. Chromatogr. A 763 295-306 (1997). 

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. 

Bergman AC, Benjamin T, Alaiya A, Waltham M, Sakaguchi K, Franzen B, et al. Identification of gel-separated tumor marker proteins by mass spectrometry. Electrophoresis 2000 21(3) 679-686. 

Albumin can be measured quantitatively by the bromcresol green method in most species (Evans and Duncan 2003). Other proteins (described below) are measured by immunometric methods. Newer methods based on proteomics technology (concentration of proteins by acetone precipitation or ultracentrifugation, separation by 2-d gel electrophoresis or chromatographic techniques with subsequent identification and quantitation by mass spectrometry) have been used experimentally (Bandara and Kennedy 2002 Chapman 2002 Thongboonkerd et al. 2002a, b). 

Mass spectrometry (MS) permits the identification of proteins by the determination of the exact mass of peptides, and the fragmentation of these peptides to determine the amino acid sequence. Protein samples, such as purified organelles or protein complexes are usually separated by their relative molecular weight on SDS-PAGE gels. The protein bands are visualized by dyes such as Coomassie brilliant blue and then are excised from the gel. The protein-containing gel slices are washed and incubated with proteolytic enzymes such as trypsin and the resultant peptides extracted from the gel-slices. 

Oiuitif and conquer, The determination of the mass of a protein by mass spectrometry often does not allow its unique identification among possible proteins within a complete proteome, but determination of the masses of all fragments produced by digestion with trypsin almost always allows unique identification. Explain. 

TAP involves a couple of cycles of purification of interacting proteins based on their affinity to another molecule bound to a matrix and the subsequent identification of interacting proteins by mass spectrometry. This is a high-throughput method, but it cannot detect weak transient interactions because such proteins with weak interactions get separated during the steps of affinity purification. The TAP method has been used to establish the interactomes or protein networks in many organisms, including yeast. A brief description of this method is presented next. 

Cao H, Deterding L J, Venable J D, et al. (2006). Identification of the antiinflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis. Biochem. J. 394 285-297. 

Eckhard Nordhoff, Anne-M. Krogsdam, Helle F. Jorgensen, Birgitte H. KaUipoMs, Brian F. C. Clark, Peter Roepstorff and Karsten Kristiansen, A rapid identification of DNA-binding proteins by mass spectrometry, Nature Biotechnology, 17 (2000), 884-888. 

The readily available experimental tools for measmement of protein expression by two-dimensional electrophoresis (2DE), and for protein identification and characterization by mass spectrometry (MS) have made a significant impact on proteonucs (Hamdan and Righetti, 2(X)5). The coupling of 2DE and MS offers an efficient tool for investigating pro-teome expression. 

In the field of proteomics, arrays can also be used to identify possible interaction partners. Here, the array is spotted with recombinant proteins or antibodies and then hybridised with labelled cell lysate or an expressed cDNA library. Additionally, techniques like two-dimensional gel electrophoresis, the identification of isolated proteins by mass spectrometry or the two-hybrid analysis are valuable tools to identify new proteins. These methods allow a large-scale study of viral and cellular proteins without knowledge of the DNA sequence (Fig. 1, IV. For review, see Pandey and Mann 2000). 

Identification of proteins by correlating the mass spectrometry data with entries in a protein or DNA database. Alternatively, the amino acid sequence of each cleaved peptide is determined by de novo methods. 

Because of the advances in the gas-phase ionization of biomacromolecules, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) has become a powerful tool for detection, identification, and structural analysis of proteins, peptides, and polynucleotides. The molecules ionized in a gas phase by these methods are subsequently analyzed by sector, quadrupole, ion-trap, or time-of-flight mass spectrometers. In particular, the MS systems consisting of ESI and triple-stage quadrupole (ESI/TSQ) or ion-trap (IT) mass spectrometry and MALDI time-of-flight (MALDl/TOF) mass spectrometry have been most widely applied to the field of protein chemistry for the accurate determination of molecular mass of proteins and peptides, determination of amino acid sequence, identification of proteins by peptide mass databases, and analysis of posttranslational modifications such as phosphorylation and glycosylation. In general, current techniques allow detenni-... 

---