Mass spectrometry protein sequencing

Protein Sequencing Mass Spectrometry Proteins were sequenced on an Applied Biosystems 477A protein sequencer. Polypeptide molecular weights were determined after analysis by a Sciex API-Ill electrospray ioniziation mass spectrometer. 

Mass spectrometry currently has assumed a central role in protein sequencing. This development has been possible with the introduction of two highly sensitive ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) and the advent of improved instrumentation capable of high-mass and high-sensitivity detection. Currently, biopolymers with a molecular mass over 100,000 Da are analyzed routinely. In the past, fast atom bombardment (FAB) [6,7] and Cf plasma desorption (PD) ionization [8] also played a limited role in protein sequencing. Mass spectrometry now has assumed... 

Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993). Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32, 1991-2002. 

Strategy followed in bottom-up sequencing proteins using mass spectrometry. 

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]. 

Figure 8.19 outlines the general strategy to characterize recombinant proteins by mass spectrometry. If the accurately measured molecular mass of the produced protein is in agreement with that calculated from the DNA sequence, then the deduced amino acid... 

Advances in mass spectrometry, in particular advances in the ionisation techniques for biomolecules, in conjunction with automation and bioinformatics, have revolutionised the process of protein sequencing and minimised the analysis times. The sequencing of proteins by mass spectrometry is discussed in more detail in section 4.3.4. 

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). 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Tandem mass spectrometry (MS/MS) produces precise structural or sequence information by selective and specific induced fragmentation on samples up to several thousand Daltons. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for sequencing by mass spectrometry. The smaller sequences can be used to deduce the sequence of the whole protein. 

Kinter, M. and Sherman, N.E., Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley, Chichester, U.K., 2000. 

The sequence of a globular protein was confirmed by a combination of enzymatic digestion and HPLC with both Fourier-transform infrared spectroscopy (LC-FTIR spectroscopy) and mass spectrometry [11]. 

Table 5.7 Theoretically predicted polypeptides from the trypsin digestion of S-lacto-globulin (/3LG) . Reprinted from J. Chromatogr., A, 763, Turula, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structure elucidation of a globular protein by particle beam liquid chromatography-Fourier transform infrared spectrometry and electrospray liquid chromatography-mass spectrometry - Sequence and conformation of /3-lactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

Assuming the sequence of the parent protein is known, it is not necessary to redetermine the whole sequence merely to locate, and sequence, that/those polypeptide(s) that have undergone modification. This can be done by examination of the total-ion-current (TIC) trace before and after protein hydrolysis for the appearance of new polypeptides or to use mass spectrometry methodology to locate those polypeptides that contain certain structural features. Examples are provided here of both methodologies. 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Gatlin, C.L., Eng, J.K., Cross, S.T., Detter, J.C., and Yates, J.R III, Automated Identification of amino acid sequence variations in proteins by HPLC/mi-crospray tandem mass spectrometry, Anal. Chem., 72, 757, 2000. 

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