Fragments, bovine serum albumin

Figure 6.4. Fragmentation spectrum of a tryptic peptide obtained from bovine serum albumin. Peptide sequence LGEYGFQNALIVR, monoisotopic [M + H]+ = 1479.796, monoisotopic [M+2H]2+ =740.402. Upper panel full scan MS spectrum. Lower panel MS/MS spectrum of a doubly-charged ion at 740.7 m/z with a ladder of y ions, the distances between which correspond to amino acid residues (upper row of letters). A shorter series of b ions is also seen (lower row of letters). See Fig. 6.5 for description of nomenclature. Note the often observed phenomenon where multiply-charged ions lose the charge during fragmentation process and, therefore, have higher m/z values than the original parent ion.

Complete sequencing of this peptide requires acquisition of additional tandem mass spectra, preferably MS3 fragmentation, of one of the low-mass y-ions. Because the peptide of interest is derived from a biological source, yet another possibility might be the use of sequence databases, similarly to the previous example. Actually, this approach works very well in this case, allowing identification of the peptide of interest as H-LGEYGFQNALIVR-OH, the 421-433 fragment of bovine serum albumin. 

Native bovine serum albumin has the surprising property of catalyzing the decomposition of the Meisenheimer complex 1,1-dihydro-2,4,6-trinitrocylohexadienate. Taylor and Silver (1976) prepared albumin fragments 1-306 and 307-581 and separated them without disulfide reduction. These were called native fragments. Neither of these fragments alone has catalytic activity to decompose the Meisenheimer complex, but when mixed together stoichiometrically, 35%... 

That conclusion is supported and extended by the virtually simultaneous publication of Teale and Benjamin (1976). These investigators studied the oxidative regeneration of bovine serum albumin, assaying the extent of refolding immunochemically. Their results showed clearly that some parts of the molecule fold faster than others. Two fragments of albumin were tested for oxidative regeneration in the same way. Substantial return of native structure was seen in both fragments. 

B) O, Anti-bovine serum albumin , anti-C-ffagment and anti-TiM-jM 9, anti-N-fragment. Reprinted, with permission, from Teale and Benjamin (1976). Copyright by the American Society of Biological Chemists, Inc. 

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.

FIGURE 4 Effect of sample preparation on the fragmentation of an rMAb observed in (A) SDS-PAGE and (B) CE-SDS with LIF detection. SDS-PAGE lanes (Lane I) molecular weight standards bovine serum albumin at (Lane 2) 8 ng and (Lane 3) 2 ng (Lane 4) rMAb control after alkylation with (Lane 5) iodoacetic acid and (Lane 6) iodoacetamide. (See color plate 4.)... 

Bovine serum albumin /9-Lactoglobulin Hemoglobin Cytochrome c Lysozyme Rat IgG and light chain fragment... 

Peters, T., Jr. and Feldhoff, R. C. 1975. Fragments of bovine serum albumin produced by limited proteolysis. Isolation and characterization of peptic fragments. Biochemistry 14, 3384-3391. 

Whey proteins contain several major allergens, such as P-lg and a-la, and minor constituents, e.g., lactoferin, bovine serum albumin (BSA), and immunoglobulins. In this fraction, proteolytic fragments of casein and fat globule membrane proteins can occur during analysis. 

A problem of the experimental measurement of local polarity in the vicinity of donor and acceptor centers incorporated into a protein (bovine serum albumin, BSA) was solved with the use of the dual fluorescence-nitroxide probe (Bystryak et al., 1986 Rubtsova et al., 1993 Fogel et al., 1994 Likhtenshtein, 1993, 1996 Likhtenshtein et al., 2001). In such a hybrid molecule, the photoactive chromophore fragment in the excited singlet state can... 

With respect to the advantage of using a protein rather than a synthetic polypeptide as a carrier, we can refer to Jaffe et who showed that an active fragment of gastrin, its C-terminal tetrapeptide amide, was antigenic when covalently attached to serum protein carriers but not when linked to poly(L-lysine) or to poly(L-glutamic acid). Walker et al. in 1973 made similar comparisons with steroid conjugates and obtained the same result, i.e., bovine serum albumin was a better carrier than poly(L-lysine). (But also see below. 

In order to determine the optimum sample preparation conditions, we initially tried several sample preparation methods in the absence of contaminants. The following peptide and protein mixture was prepared (peptides and protein were purchased from Sigma Chemicals, St. Louis, MO) 1 pmol/jul Parathyroid Hormone Fragment 39-68 (PHF), MW = 3285.7, 10 pmol/ul Pigeon Heart Cytochrome C (PHC), MW = 12,173, and 10 pmol/ul Bovine Serum Albumin (BSA), MW = 66,256. A saturated solution of Sinapinic acid in 1 1 ethanol water was used as matrix. 

Fig 1. 1 pmol Parathyroid Hormone Fragment 39-68 (PHF), lOpmoI Pigeon Heart Cytochrome C (PHC), 10 pmol Bovin Serum Albumin (BSA) in saturated sinapinic acid matrix, desorbed from a. Polyethylene Membrane 113-2 b. Polypropylene Membrane 1222 c. Type 61 Disposable IR Card d. C8 Extraction Disk e. C18 Extraction Disk. 

Coupling of F(ab ) fragments to amino-groups on bovine serum albumin-cellulose acetate membranes using succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate, and attachment to platinum electrodes human IgG [6]... 

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