Protein Sequencing by Mass Spectrometry

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. 

Joubert-Caron R et al. Protein analysis by mass spectrometry and sequence database searching a proteomic approach to identify human lymphoblastoid cell line proteins. Electrophoresis 2000 21 2566— 2575. 

Conformational isomers of OBPs that are detectable by gel electrophoresis have also been found in three species of scarab beetles (Deyu and Leal, 2002 Wojtasek et al., 1999). We have demonstrated by protein sequencing and mass spectrometry that these electrophoretically distinct bands are from proteins with the same primary sequences (Deyu and Leal, 2002 Wojtasek et al., 1999). Although the term isoform means different forms of a protein it has been... 

Standing, K.G. (2003) Peptide and protein de novo sequencing by mass spectrometry. 

Fig. 5. The approach to probing higher order protein structure by mass spectrometry is shown. The cleavage of a hypothetical protein generates fragments (a, b, c, a+b, and b+c) that can be monitored by MALDI-MS. Here, arrows denote possible cleavage sites for proteolysis. The adduction of this protein with another to form a complex leads to a suppression of proteolysis in the region of association as well as fragments from the other protein (xf). An example is shown for the limited proteolysis of p21-B/Cdk2 complex. The suppression of proteolysis in a 24 residue region of the sequence helps identify this area of association...

A. Tsarbopoulos, B. N. Pramanik, J. Labdon, P. Reichert, G. Gitlin, S. Patel, V. Sardana, T. L. Nagabhushan, and P. P. Trotta, Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) confirmation of the GM-CSF amino acid sequence by mass spectrometry. Protein Sci. 2 (1993), 1948-1958. 

Gray [192] proposed a method in which by acetylating the initial protein the terminal peptide liberated by specific enzymic degradation was isolated and its permethylated derivative sequenced by mass spectrometry. 

In this technology, the cell or tissue preparation is incubated with the beads to allow specific binding of the kinases, and then the beads are washed extensively under non-denaturing conditions to remove nonspecific proteins. The specifically bound kinases (and kinase binding partners) are eluted under denaturing conditions, subjected to proteolysis to liberate constituent peptides, and the latter are subsequently analyzed and sequenced by mass spectrometry methods. 

Joubert-Caron, R., Le Caer, J.P., Montandon, F., Poirier, F., Pontet, M., Imam, N., Feuillard, J., Bladier, D., Rossier, J., and Caron, M., 2000, Protein analysis by mass spectrometry and sequence database searching a proteomic approach to identify human lymphoblastoid cell line proteins [In Process Citation]. Electrophoresis 21 2566-2575. 

The most common proteomics approach uses two-dimensional gel electrophoresis to separate cellular proteins, followed by in-gel tryptic digestion of the protein spots and identification of the peptide sequences by mass spectrometry. There are s ificant problems with this approach, which so far, have limited its usefulness for drug taiget discovery. A number of protein classes such as int ral membrane proteins, positively chaiged and hydrophobic proteins are difficult to separate. There are often selective losses of individual protdns, for reasons that are not well understood, so the claim for a global picture is not entirefy accurate. The approach is semi-quantitative at best. Importantly, low abundance proteins may not be detected. However, newer approaches are constantly being developei Approaches which... 

Figure 6. Tandem mass spectrometry for protein sequencing. One peptide (2826Da) from the Mus spretus peptide mixture (Figure 5) was selected for sequencing by mass spectrometry. When the peptide ion is fi mented in the mass spectrometer, a complex pattern of ions are obtained, corresponding to the successive loss of amino acids from one end or the other of the peptide. From the mass differences between the successive ions, it is possible to reconstruct the peptide sequence of the protein. (J = leucine / isoleucine)...

Figure 3 Protein identification by mass spectrometry, in a typicai strategy, digested peptides are anaiyzed by MALDi-TOF-MS in order to determine the masses of intact peptides. These masses can be used in correiative database searches to identify exact matches. If this approach fails, ESI-MS/MS analysis can be used to generate peptide fragment ions. These can be used to search less robust data sources and to produce de novo peptide sequences. (Reproduced with permission from Twyman RM (2004) Principles of Proteomics. Abington, UK Bios/Garland Publishers.)...

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

It should be emphasized that the nature of all presented protocols is very general and, thus, their application for a comprehensive characterization of your favorite multiprotein complex (YFMPC) in yeast might require only minor modifications. The logical sequence of all required steps is schematically shown in Fig. 2.1. The initial large-scale Ni affinity isolation of eIF3 followed by mass spectrometry (MS) of its subunit composition has already been described (Asano et al, 2002), and methods for identification of protein-protein interactions such as yeast two-hybrid (Y2H) and in vitro glutathione-S-transferase (GST) pull-down analysis are presented in volume 429. This chapter focuses on a description of the small-scale one-step in vivo affinity purification techniques that were used to determine the effects of deletions and... 

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