Protein mass spectrum

Snyder, A.P., Interpreting Protein Mass Spectra A Comprehensive Resource, American Chemical Society, Washington, D.C., 2000. 

C. Fenselau, MALDI MS and Strategies for Protein Analysis, Anal. Chem. 1997,69, 661 A R. W. Nelson, D. Nedelkov, and K. A. Tubbs, Biomolecular Interaction Analysis Mass Spectrometery, AnaL Chem. 2000, 72, 405A J. J. Thomas, R. Bakhtiar, and G. Siuzdak, Mass Spectrometry in Viral Proteomics, Acc. Chem. Res. 2000,33, 179 A. P. Snyder, Interpreting Protein Mass Spectra (Washington, DC American Chemical Society, 2000) S. C. Moyer and R. J. Cotter, Atmospheric Pressure MALDI, Anal. Chem. 2002, 74, 469A. 

The use of perfusion colunms (Ch. 1.4.1) was also evaluated to speed up the LC separation of proteins prior to ESI-MS [49]. Five-fold faster analysis was reported. Due to the narrow chromatographic peaks (5-10 s), the number of protein mass spectra available for transformation is limited. 

A. P. Snyder, Interpreting Protein Mass Spectra, Oxford University Press, Oxford, UK, 2000. C. Dass. Principles and Practice of Biological Mass Spectrometry, Wiley-Interscience, New York, 2001. 

A. P. Snyder, Interpreting Protein Mass Spectra, Oxford University Press, Oxford, UK,... 

The first millisecond protein HX measurements were made by automated quench-flow pulse labeling and were aimed at characterizing early protein-folding intermediates [15, 16]. In these experiments, unlabeled protein was mixed with D O and incubated for a short period (ms), followed by a rapid pH drop and flash freeze to quench the reaction. Labeled samples were then analyzed by NMR (this was 1988, the same year that John Fenn showed the first electrospray protein mass spectra at the American Society for Mass Spectrometry meeting). Quench-flow HX for protein folding was translated to MS a few years later by Miranker and coworkers [17]. 

The importance of linked scanning of metastable ions or of ions formed by induced decomposition is discussed in this chapter and in Chapter 34. Briefly, linked scanning provides information on which ions give which others in a normal mass spectrum. With this sort of information, it becomes possible to examine a complex mixture of substances without prior separation of its components. It is possible to look highly specifically for trace components in mixtures under circumstances in which other techniques could not succeed. Finally, it is possible to gain information on the molecular structures of unknown compounds, as in peptide and protein sequencing (see Chapter 40). 

A typical electrospray analysis can be completed in 15 min with as little as 1 pmol of protein. An analysis of the cord blood of a baby (Figure 40.6) showed quite clearly that five globins were present, viz., the normal ones (a, (3, Gy, and Ay) and a sickle-cell variant (sickle (3). The last one is easily revealed in the mass spectrum, even at a level of only 4% in the blood analyzed. 

A sample of the protein, horse heart myoglobin, was dissolved in acidified aqueous acetonitrile (1% formic acid in HjO/CHjCN, 1 1 v/v) at a concentration of 20 pmol/1. This sample was injected into a flow of the same solvent passing at 5 pl/min into the electrospray source to give the mass spectrum of protonated molecular ions [M + nH] shown in (a). The measured ra/z values are given in the table (b), along with the number of protons (charges n) associated with each. The mean relative molecular mass (RMM) is 16,951,09 0.3 Da. Finally, the transformed spectrum, corresponding to the true relative molecular mass, is shown in (c) the observed value is close to that calculated (16,951.4), an error of only 0.002%. 

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. 

FIGURE 5.23 Electrospmy mass spectrum of the protein, aerolysin K. The attachment of many protons per protein molecule (from less than 30 to more than 50 here) leads to a series of m/z peaks for this single protein. The inset shows a computer analysis of the data from this series of peaks that generates a single peak at the correct molecular mass of the protein. (Adapted from Figure 2 in Mann, M., and Wilm, M., 1995. Trends in Biochemical Sciences 20 219-224.)... 

Figure 12.9 MALDI-TOF mass spectrum of chicken egg-white lysozyme. The peak at 14,307.7578 daltons (amu) is due to the monoprotonated protein, M+H+, and that at 28,614.2188 daltons is due to an impurity formed by dimerization of the protein. Other peaks are various protonated species, M+H rH ...

If, however, we consider a protein of modest size, such as aprotin with a molecular formula of C284H432N84O79S7 at a similar mass spectrometer resolution, the molecular-ion region of its mass spectrum, shown in Figure 4.14, does not show the individual isotopic contributions, a resolution around 5000 being required for these to be evident (Figure 4.15). 

Figure 5.19 MALDI-ToF mass spectrum, providing a molecular-weight profile of the tryptic peptides derived from spot 22 (see Figure 5.18) of the silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaUddnen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, copyright 2001. John Wiley Sons Limited. Reproduced with permission.

Figure 3.9 MALDITOF mass spectrum showing over-expressed soluble core domain protein of cytochrome B5.

Note that the dominant peak in the observed mass spectrum is from the overexpressed protein. The observation of several ribosomal proteins allows for an internal calibration to be performed on the mass spectrum, greatly improving the mass measurement accuracy. 

Because online separations provide such a wealth of information about target proteins, interpretation becomes of critical importance in order to make full use of the data. The first step in any analysis of LC-MS data involves integration and deconvolution of sample spectra to determine protein mass and intensity. In manual analysis (Hamler et al., 2004), users identify protein umbrellas, create a total ion chromatogram (TIC), integrate the protein peak, and deconvolute the resulting spectrum. Deconvolution of ESI spectra employs a maximum entropy deconvolution algorithm often referred to as MaxEnt (Ferrige et al., 1991). MaxEnt calculates... 

The central engine of this data workflow is the process of spectral deconvolution. During spectral deconvolution, sets of multiply charged ions associated with particular proteins are reduced to a simplified spectrum representing the neutral mass forms of those proteins. Our laboratory makes use of a maximum entropy-based approach to spectral deconvolution (Ferrige et al., 1992a and b) that attempts to identify the most likely distribution of neutral masses that accounts for all data within the m/z mass spectrum. With this approach, quantitative peak intensity information is retained from the source spectrum, and meaningful intensity differences can be obtained by comparison of LC/MS runs acquired and processed under similar conditions. 

Then the mass spectrum of the mentioned peptide mixture is measured. The set of molecular mass values (peak list) corresponding to individual peptides is characteristic for the protein and can be considered as its fingerprint. 

Figure 6.7 Mass spectrum obtained from sample 4. The peaks corresponding to milk proteins are labelled...

Proteins and peptides are most often seen in the mass spectra as pseudomolecular ions, that is, molecules with attached charge-carrying protons (in the negative-ion mode, proteins and peptides lose protons and thus acquire a negative net charge). This additional proton has to be taken into consideration in order to predict correctly the m/z value at which the peptide of interest will be seen in a mass spectrum. For example, a peptide whose molecular weight (MW) (or molar mass) is equal to 2000 Da, when singly ionized, will be detected at 2001 m/z (for simplification, we assume the mass of proton as equal to 1) ... 

R. Craig, et al., Using Annotated Peptide Mass Spectrum Libraries for Protein Identification. J. Proteome Res., 5, no. 8 (2006) 1843-1849. 

Two adjacent signals in the ESI mass spectrum of a pure protein have mlz values of 1428.6 and 1666.7, respectively. Since adjacent signals differ in the value of their respective charge states by 1, the following expressions can be solved for the value of one of the charge states (z2 in this case) and then for the molecular mass of the protein analyte, m. 

Two adjacent signals in an electrospray mass spectrum of a pure protein sample have m/z values of 893.9 and 834.3. Calculate the charge state of each signal and the mass of the neutral protein. (+14 and +15 12,500 Da). 

Figure 14.6 Representative mass spectrum showing ladder of a bisglycosylated glycopeptide obtained from screening of a glycopeptide library for the mannose binding protein. The nonanoic acid (Non) and tridecanoic acid (Tri) encode for Ser(Man) (Sm) and Asn(Man) (Nm), respectively.

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