Proteins mass measurement

The accuracy of protein mass measurement depends on a number of factors because many proteins are sufficiently massive that resolution is not achieved from additional species such as salts or matrix adducts. Under these conditions, such species cause peak broadening and result in a small shift in measured mass to higher values. Post-translational modifications such as glycosylation can often be resolved for the smaller proteins such as ribonuclease B (Figure 3) with a single glycosylation site, but larger compounds with multiple modifications often produce only broad peaks. 

Another type of ion is formed almost uniquely by the electrospray inlet/ion source which makes this technique so valuable for examining substances such as proteins that have large relative molecular mass. Measurement of m/z ratios usually gives a direct measure of mass for most mass spectrometry because z = 1 and so m/z = m/1 = m. Values of z greater than one are unusual. However, for electrospray, values of z greater than one (often much greater), are quite coimnonplace. For example, instead of the [M + H]+ ions common in simple Cl, ions in electrospray can be [M + n-H]- where n can be anything from 1 to about 30. 

The use of CIEF in combination with FTICR has been demonstrated in an analysis of the E. coli proteome (Jensen et al., 1999). For these experiments, E. coli was grown in a medium depleted of rare isotopes in order to increase the mass measurement accuracy. The high abundance isotopes are present at approximately 98.89% 12C, 99.63% 14N and 99.985% H. For peptides, the presence of rare isotopes does not significantly change the spectra but with undigested proteins, mass accuracy can be limited by the broadened distribution of ions of any given protein due to the incorporation... 

Clauser, K. R. Baker, P Burlingame, A. L. Role of accurate mass measurement (+10ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal. Chem. 1999,71,2871-2882. 

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. 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

Clauser, K.R. Baker, P. Burlingame, A. Role of Accurate Mass Measurement ( 10 Ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching. Anal. Chem. 1999, 71, 2871-2882. 

Example ESI on a magnetic sector instrument set to R = 20,000 allows for the flail resolution of isotopic peaks in case of medium-molecular weight proteins (Fig. 11.21). This enables the direct determination of the charge state of the ions from the spacing of the isotopic peaks, i.e., 1 1h- for the lysozyme ion due to the average spaces of Am = 0.091 u and 13h- for the myoglobin ion due to Am = 0.077 u. In this particular case, the lysozyme [M+llH]" ion serves as a mass reference for the accurate mass measurement of the unknown" [M+13H] ion. [103]... 

Since FTMS provides high resolution and high mass accuracy, it enables detailed structural investigation for small molecules and proteins as well. There has been a publication examining the effectiveness of accurate mass measurements in minimizing false protein... 

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