Proteins electrospray mass spectra

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 3.11 Electrospray mass spectrum of apo-myoglobin and the deconvoluted data giving the molecular mass of the protein. 

Figure 6 Electrospray mass spectrum of the protein myoglobin, FW 16950. The numbers at the peaks denote the number of charges on the ions.

Fig. 3 LCMS Analysis of Fusion rCRALBP (A) RP-HPLC ultraviolet profile ( 220nm) froHi analysis of 1.5 (ig fusion human rCRALBP on a 50- X Vydac C18 column (lx 250 mm) at 50 lL/min usmg the indicated gradient. Solvent A was 0 1% trifluoracetic acid (TFA) and solvent B was 84% acetonitrile, containing about 0.07% TFA. (B) Reconstructed total ion current from electrospray-mass spectral analysis of the fusion rCRALBP liquid chromatography shown in A. (C) Electrospray-mass spectrum of fusion human rCRALBP indicating the presence of a protein of = 39,114 4 (calculated = 39,110). The deconvoluted spectrum is shown in the inset.

Fig 4 LCMS Analysis of Non-Fusion rCRALBP (A) RP-HPLC ultraviolet profile, (B) reconstructed total ion current, and (C) electrospray mass spectrum with the deconvoluted spectrum shown m the inset LCMS analysis of nonfusion human rCRALBP (1.5 p,g) was performed as described in the legend to Fig. 3, yielding a measured protein mass of = 36,347 4 (calculated = 36,343). 

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 1.11. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrum of a glu-c-digested 191-kDa protein, collected on the NHMFL 9.4-T system. (Inset) A resolved 30-kDa fragment. Data kindly provided by N. Kelleher.

Although this technique was introduced in early 1990s, it was the introduction of a MALDI-TOF instrument capable of 50 ppm mass accuracy that made PMF a routine procedure. In MALDI-TOF mass spectrometry, peptides appear as singly charged species in the mass spectrum (Figure 4.4-1). Unlike an electrospray (ESI) mass spectrum, which displays multiply charged species, the MALDI-TOF spectrum is simple to interpret. PMF can also be used to identify proteins in ESI spectra, but it is seldom used because the peptide masses would need to be decon-voluted for each search. 

Fig. 3.14. Electrospray ionization mass spectrum of bovine albumin protein (A4= 66,300), illustrating the ability of electrospray ionization to produce multiply charged, large biomolecular ions. Reprinted from Smith et al. (1990) with permission from the American Chemical Society.

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