Mass spectrometry tryptic digest

Table 5.8 Polypeptides detected during the LC-electrospray-MS analysis of the tryptic digest from / -lactoglobulin (/ILG). Reprinted from 7. Chromatogr., A, 763, Tnrnla, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structnre elncidation of a globular protein by particle beam hqnid chromatography-Fourier transform infrared spectrometry and electrospray hqnid chromatography-mass spectrometry - Seqnence and conformation of / -lactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

Figure 5.27 Selective detection of lactolated peptides from a tryptic digest of / -lacto-globulins by LC-electrospray-MS-MS, showing (a) the total-ion-cnrrent trace in full-scan mode, and (b) the total-ion-current trace in neutral-loss-scanning mode. Figure from Selective detection of lactolated peptides in hydrolysates by liquid chromatography/ electrospray tandem mass spectrometry , by Molle, D., Morgan, F., BouhaUab, S. and Leonil, J., in Analytical Biochemistry, Volume 259, 152-161, Copyright 1998, Elsevier Science (USA), reproduced with permission from the publisher.

Wahl, J.H., Gale, D.C., Smith, R.D. (1994). Sheathless capillary electrophoresis-electrospray ionization mass spectrometry using 10 pm I.D. capillaries analyses of tryptic digests of cytochrome c. J. Chromatogr. 659, 217-222. 

Groseclose M, Anderson M, Hardesty W, et al. Identification of proteins directly from tissue in situ tryptic digestions coupled with imaging mass spectrometry. J. Mass. Spectrom. 2007 42 254-262. 

Xue, Q., Dunayevskiy, Y. M., Foret, F., and Karger, B. L. (1997). Integrated multichannel microchip electrospray ionization mass spectrometry analysis of peptides from on-chip tryptic digestion of melittin. Rapid Commun. Mass Spectrom. 11, 1253 — 1256. 

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. 

Li, M. X. Wu, J. T. Parus, S. Lubman, D. M. 1998. Development of a three-dimensional topographic map display for capillary electrophoresis/mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector applications to tryptic digests of isoforms of myelin basic protein. ./. Am. Soc. Mass Spectrom., 9,701-709. 

FIGURE 1 Example of a gel-free-oriented proteomics nano-LC/MS-MS workflow in which bacterial culture proteins digested to tryptic peptides are separated via LC and peptides subsequently analyzed by mass spectrometry. In the process, the spectrometer rapidly cycles every few seconds and examines a size window in which peptide-derived MSI ions are analyzed to define MS/MS (MS2) spectra. The MS/MS (MS2) spectrum generated for each peptide then enters a bioinformatic pipeline for sequence identification, statistical validation, and quantification. 

Tryptic Maps of Relaxin and Relaxin B-chain. Digestion of the A-chain of human relaxin with trypsin can theoretically result in the release of five fragments that of the B-chain in the release of six fragments as illustrated in Table II. A typical tryptic map of relaxin B-chain is shown in Figure 2. The peptide was reduced and carboxymethylated with iodoacetic acid before enzymatic digestion. The peptide assignments were made after analysis of the peaks by amino acid hydrolysis for amino acid composition and confirmed by fast atom bombardment mass spectrometry (FAB-MS) as shown in Table IH... 

Recombinant human growth hormone, tryptic digests Vydac 218TPB5 (Cl8), 5 pm, 300 A A 0.1% TFA/water, B 0.09% TFA/acetonitrile, gradient from 0 to 60% B 250 mm x 100 pm i.d. Electrically assisted capillary HPLC, coupling with electrospray ionization-mass spectrometry... 

Binding of sarin and soman to a tyrosine residue present in blood has been observed by Black et al. (51) When sarin or soman was incubated with human plasma, phosphonylated tyrosine was observed by LC/MS after Pronase digestion, in addition to phosphonylated serine. The precise site of this residue has not yet been confirmed but it is associated with the albumin fraction. A phosphonylated tryptic peptide [/-PrO(CH3)P(0)]-Tyr-Thr-Lys, consistent with albumin, has been identified but this sequence is also present in other proteins. Before the advent of modem mass spectrometry, diisopropyl fluorophosphate was reported to bind... 

Another approach to selenized yeast is to characterize Se-containing proteins. The application of polyacrylamide gel electrophoresis (PAGE) has been proposed with the introduction of the protein spot to ICP-MS via LA or ET atomization [62, 93]. The feasibility of matrix-assisted laser desorption ionization (MALDI)D timeDof-Bight mass spectrometry (TOF-MS) prior to ES tandem MS as applied to tryptic protein digests has also been explored [135, 136]. 

This example not only demonstrates how mass spectrometry is a powerful tool in the analysis of proteins, but it also sets a good example of how the different mass spectrometric techniques should be combined to give the best answers possible. The molecular mass was determined by the more accurate ESI-MS analysis, and the initial tryptic digest was studied by exploiting the ability of ESI-MS to be directly interfaced with an HPLC. Then MALDI-TOF MS was used efficiently to obtain the MS-MS (PSD) data. 

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