Albumin, tryptic digest

Cytochrome c, bovine, tryptic digest, chicken albumin, tryptic digest Vydac Cl8, 3 pm Acetonitrile-0.07% TFA in water (gradient from 0 100 to 40 60) 60 mm x 180 pm i.d. 99... 

Fast and simple 2D HPLC was also shown to be effective for the separation of a tryptic digest of bovine serum albumin (BSA) (Kimura et al., 2004). Every... 

FIGURE 12.5 Human serum tryptic digest analysis. Fractionation in the first LC dimension was performed using a C18 column at pH 10. Fractions were analyzed using NanoEase 0.3 x 150 mm Atlantis d18 column. Approximately 66 lg (400 pmole of semm albumin peptides) was injected on column. Arrow points to a selected albumin peptide illustrating a local column mass overloading. Ten-5mm wide fractions were collected in 1st LC dimension. 

Recently, sulfur mustard has been shown to alkylate a cysteine residue in human serum albumin (10). The site of alkylation was identified in a tryptic digest of albumin from blood exposed to [14C]sulfur mustard. A sensitive method for its analysis was developed based on Pronase digestion of alkylated albumin to the tripeptide S-[2-[(hydroxyethyl)thio]ethyl-Cys-Pro-Phe, and detection using micro-LC-MS-MS. In vitro exposure of human blood to > 10 nM sulfur mustard could be detected employing this method. The analytical procedure was successfully applied to albumin samples from Iranian casualties of the Iraq-Iran war. 

Example The reduced sample consumption of nanoESI allows for the sequencing of the peptides (Chap. 9.4.7) obtained by tryptic digestion of only 800 fmol of the protein bovine semm albumin (BSA, Fig. 11.6). [66] The experiment depicted below requires each of the BSA-derived peptide ions in the full scan spectrum to be subjected to fragment ion analysis by means of CID-MS/MS on a triple quadrupole instmment (Chaps. 2.12 and 4.4.5). 

Fig. 11.6. Peptide sequencing by nanoESI-CID-MS/MS from a tryptic digest of bovine serum albumin (BSA) 800 fmol of BSA were used, (a) Eull scan spectrum, (b) fragmentation of the selected doubly charged peptide ion at m/z 740.5. Adapted from Ref. [66] by permission. Nature Publishing Group, 1996.

Two-dimensional SECxRP can be also used for protein and peptide separation, and this approach has been used for separation of peptides in tryptic digests of ovalbumin and serum albumin [14],... 

A method using micro-LC/tandem MS was developed for analysis of the tryptic digest containing the intramolecular albumin lysine-lysine adduct, which enabled the detection of exposure of human blood to > 1 xM phosgene in vitro. The method has not yet been applied to animal or human samples. 

Figure 12. Product ion spectrum of molecular ion [M + 2H]2+ (m/z 862.0) of 0=14C-(T25-T28) in a tryptic digest of albumin isolated from human blood that was exposed to [14C]phosgene (reprinted with permission from Noort, D. et al., Chem. Res. Toxicol., 13, 719-726 (2000). Copyright (2000) American Chemical Society...

Figure 5. Reverse phase HPLC of in gel tryptic digests of 25 pmol transferrin (A) and 10 pmol bovine serum albumin (B) and of the corresponding digests carried out on blank sections of gels (lower profiles shown in above two figures). In each instance 90% of the digest was subjected to HPLC on a 1.0 mm ID Vydac C-18 coluttm eluted at 50 pl/min. The respective full scale deflections were 18.9 mV for panel A and 4.4 mV for panel B with 0.5 volt corresponding to an absorbance of 1.0 at 210 nm.

The high resolving power of FT-ICR-MS can readily be exploited in bottom-up protein identification. A nice example is the identification of high-abundant proteins in a tryptic digest of human plasma without any prior separation. The 2745 peaks in the spectmm could be reduced to 1165 isotopic clusters and 669 unique masses, 82 of which matched tryptic fragments of albumin (93% sequence coverage) and 16 others transferrin (41%) [39]. The same group showed that a theoretically predicted retention time of a tryptic peptide can be applied as an additional protein identification tool, next to its accurate mass acquired in LC-FT-ICR-MS. [40-41]... 

Nerve agents also react with a tyrosine residue associated with the albumin fraction in blood (Black et al, 1999) (Figure 10). Analysis of tryptic digests from plasma incubated with sarin identified a phosphylated tripeptide, MeP(0)(0 Pr)-Tyr-Thr-Lys, consistent with the protein being albumin (tyrosine residue 411), although this sequence is common and occurs in other proteins. Before the advent of modern mass spectrometry, diisopropyl fluorophosphate was reported to... 

The spectrophotometric evidence reviewed above for the binding of a proportion of the phenolic hydroxyl groups of the tyrosine residues of native proteins is supported by work on the action of tyrosinase on proteins. Sizer (1946) found that this enzyme oxidizes the tyrosine residues in native trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Native ovalbumin, human and bovine serum albumin, tobacco mosaic virus (nucleoprotein), human y- and bovine /3-globulins, and bovine fibrinogen are not susceptible to tyrosinase, but become so after tryptic digestion. It was shown (Sizer, 1947) that for the proteins which are oxidized by tyrosinase in the native state, the observed reaction does indeed occur with the intact proteins and does not require preliminary degradation to tyrosine peptides or free tyrosine. The kinetics of the oxidation of tyrosine by tyrosinase have been studied spectropho-tometrically (Mason, 1948 etc.). 

In tube No. i without serum, 8 mm. of albumin were digested with, o.i c.c. of serum, the anti-tryptic action is already manifest, and it gradually increases with the quantity of serum in the presence of 1 c.c. of serum the tryptic action is completely arrested. The serum obtained during fasting is less active than after a meal. Further, the anti-proteolytic power of dog serum... 

FIGURE 47.6 Schematic diagram of the microfluidic device fabricated from cyclic olefin copolymer containing poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) monolith (top) and chromatogram of tryptic digested bovine serum albumin in the reversed-phase mode. Conditions digest sample 5 pmol/ xL, mobile phase A 5% acetonitrile in 0.1% aqueous TFA, mobile phase B 70% acetonitrile in 0.1% aqueous TEA, gradient 0% B for 5 min, then from 0% to 70% B in 60 min, flow rate 300 nL/min. 

The performance of the device was demonstrated with the separation of a tryptic digest of bovine serum albumin (BS A) in the reversed-phase mode using a gradient of acetonitrile in aqueous solution of trifiuoroacetic acid as the mobile phase. The separated peptides were detected by mass spectrometer. Figure 47.6 shows both the device and separation of a sample containing 5 pmol of peptides. Comparison with a database revealed an excellent amino acid sequence coverage of 70%. 

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