Myoglobin tryptic peptides

FIGURE 19.6 Representative chromatograms for a myoglobin tryptic peptide, LFTGHPETLEK, obtained from (a) human plasma blank and (b) myoglobin spiked in human plasma, and for a somatropin tryptic peptide, LFDNAMLR, obtained from (c) human plasma blank and (d) somatropin spiked in human plasma. [Figure (a) and (c) reprinted from Li et al. (2009) with permission of Rapid Communication Mass Spectrometry.]... 

As an example. Figure 2.1A shows an MS spectrum of [M -H 2H] ion at m/z 714.4 of HNE-modified apo-myoglobin tryptic peptide, LFTGH PETLEK, where the asterisk indicates His modification as a Michael adduct. Figure 2.1B shows the MS/MS spectrum of the... 

Figure 1. Concentration removal of Urea from myoglobin tryptic digest prior to microbore LC/MS using a Michrom Peptide Trap Cartridge. Sample contains 100 pmol of digest dissolved in lOOOjul of2 M Urea. Sample is run on a 1 x 150 mm RC-18 column.

Comparative HPLC peptide maps of transferrin and myoglobin were used to evaluate relative yields of tryptic peptides from different types of blotting membranes. To ensure identical transfer conditions, replicate lanes from a single gel were transferred to side-by-side strips of nitrocellulose, Immobilon P PVDF and Trans-Blot PVDF as described in Methods. As shown in Fig. 1, our results confirm the observations of Fernandez et al. (15) that optimal... 

The use of tryptic peptides derived from related proteins, e.g., horse myoglobin in the analysis of human myoglobin, as IS in targeted quantitative analysis has been proposed as well [116]. 

As noted earlier, the mass range of reflectrons is limited to an upper limit of -5 kDa. This mass is usually sufficient for the analysis of tryptic peptides. At masses >5 kDa, a linear detector must be used and resolution drops rapidly to <100, with peaks becoming very broad. The bases of the peaks are several hundred mass units wide even for small proteins, such as myoglobin at mk 16,950, (illustrated in Figure 2.20), and the accuracy of mass measurement is only -0.1%. 

Recently, accelerated trypsin digestion was achieved in the presence of organic solvent for protein quantitation in complex matrix (i.e., human plasma) (Li et al., 2009). Myoglobin was used as the model protein and somatropin was served as the analog protein internal standard. The digestion was accomplished in 30 min in human plasma, and 50% methanol (by volume) was found to be optimal in terms of tryptic peptide yield for this assay (Fig. 19.4 Li et al., 2009). The increase in the enzymatic reaction rate with the addition of organic solvent likely resulted from the partial denaturation of the substrates, which allows trypsin quick access to cleavage sites that are otherwise buried deep within the... 

In a separate study. Rill and Al-Sayah examined two-dimensional electrophoresis of myoglobin tryptic polypeptides(47). The first dimension used a cross-linked polyacrylamide gel as the support medium. Advantage was then taken of the liquid nature of the cold triblock copolymer solutions to eliminate interface issues the cross-linked gel was simply pushed into the triblock system. With few exceptions, after the second dimension of electrophoresis, most peptides lie on a diagonal line, indicating that the predominant separation mechanism is the same in both media Al). 

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