Protein digestibility disulfide bonding

Zhang, H.M., McLoughhn, S.M., Frausto, S.D., et al. (2010) Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry. Analytical Chemistry, 82 (4), 1450-1454. 

Peptide map analysis of a protein containing two or more cysteine residues typically employs reduction and alkylation chemistry for efficient, reliable proteolysis, reproducible chromatographic profiles, and straightforward characterization by MS. However, when protease digestion is carried out on a nonreduced protein, the disulfide bonds in the protein will maintain the covalent linkage between the peptides that are involved in the disulfide bond. In many cases, it is then possible to choose a protease that will ensure one cysteine residue in each peptide upon cleavage, such that each disulfide bond will associate with a pair of proteolytic peptides. 

The fact that a denatured protein can spontaneously return to its native conformation was demonstrated for the first time with ribonuclease, a digestive enzyme (see p. 266) consisting of 124 amino acids. In the native form (top right), there are extensive pleated sheet structures and three a helices. The eight cysteine residues of the protein are forming four disulfide bonds. Residues His-12, Lys-41 and His-119 (pink) are particularly important for catalysis. Together with additional amino acids, they form the enzyme s active center. 

The protected protein was deprotected with HF and then with mercury(II) acetate in 50% AcOH to remove the Acm groups. Oxidative folding gave the desired peptide. A tryptic digest was separated on HPLC and the seven identified peaks confirmed the correct disulfide bonds in the product. 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

Schematic diagrams of the amino acid sequences of chymotrypsin, trypsin, and elastase. Each circle represents one amino acid. Amino acid residues that are identical in all three proteins are in solid color. The three proteins are of different lengths but have been aligned to maximize the correspondence of the amino acid sequences. All of the sequences are numbered according to the sequence in chymotrypsin. Long connections between nonadjacent residues represent disulfide bonds. Locations of the catalytically important histidine, aspartate, and serine residues are marked. The links that are cleaved to transform the inactive zymogens to the active enzymes are indicated by parenthesis marks. After chymotrypsinogen is cut between residues 15 and 16 by trypsin and is thus transformed into an active protease, it proceeds to digest itself at the additional sites that are indicated these secondary cuts have only minor effects on the enzymes s catalytic activity. (Illustration copyright by Irving Geis. Reprinted by permission.)...

Reduction of disulfide bonds followed by alkylation of the free cysteines to prevent re-oxidation, while not essential for the digestion of most proteins, generally gives better results. This is due to increased susceptibility of the reduced/ alkylated protein to tryptic digestion and the absence of any disulphide-linked peptides (which are not matched in the database search) from the PMF data (6). 

It is evident from the discussions above that mass spectrometric method in combination with enzymatic digestion offers a convenient approach to the characterization of GM-CSF and its variants. ESI-MS method demonstrated a mass accuracy of better than 0.01% for a recombinant protein. The mass spectral data of the enzymatic digest of GM-CSF and its variants allow the precise determination of the molecular weights of the peptides, leading to the identification of sites of covalent modifications, the disulfide bonding pattern, and confirmation of the cDNA-derived sequence of the protein. 

To verify that Iso-4 was interconvertible with IFN-a-2b, a sample of purified Iso-4 was treated under mild acidic conditions in an attempt to convert it to IFN-a-2b. The MW measurement of the converted protein by RP-HPLC/ESI-MS confirmed that Iso-4 could be converted to IFN-a-2b under mild acidic conditions. Furthermore, the IFN-a-2b obtained from the conversion of Iso-4 was enzymatically digested with trypsin and studied by RP-HPLC/ESI-MS to assess the status of disulfide bonds. The presence of the two disulfide-bonded peptide fragments, Ti-ss-Tio and Ts-ss-Tn, revealed the correctly folded IFN-a-2b. 

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