Laser Desorption Mass Spectrometry in Protein Analysis - American

Chapter 12

Laser Desorption Mass Spectrometry in Protein Analysis Off and On Membranes

Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: December 21, 1993 | doi: 10.1021/bk-1994-0549.ch012

Martha M . Vestling Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, M D 21228

Because matrix-assisted laser desorption mass spectrometry (LDMS) provides considerably more accurate molecular weights than the important well-established and widely used protein technique, gel electrophoresis, several LDMS instruments have recently become commercially available. The short analysis time, ease of operation, and price of these instruments is changing the way proteins are characterized. This report describes several ways LDMS can be used to analyze proteins and peptides. First the utility of the technique will be illustrated with data from cyanogen bromide and tryptic digests. Second, acquisition of laser desorption spectra of proteins and peptides absorbed on membranes will be described. And lastly, some guidelines for preparing proteins and peptides for LDMS will be presented. Analysis of Cyanogen Bromide Digests and Mass Balance Strategy Cyanogen bromide reacts with protein methionine residues in the presence of acid and leads to cleavage of the protein chain at the C-terminal of each methionine. Since proteins usually contain only a few methionine residues, cyanogen bromide digestion produces only a few peptides. By determining the molecular weights of the peptides, a protein with a predicted sequence can be searched or mapped for unexpected features. Two different amino acid sequences in the literature for the protein Staphylococcus aureus V8 protease made it a candidate for mapping (7, 2). S. aureus V8 protease itself is widely used to generate peptides for sequencing, since it catalyzes the hydrolysis of amide bonds whose carbonyls are contributed by glutamic acid residues. The molecular weight of S. aureus V8 protease was determined by LDMS to be 29768 ± 300 (3), which was considerably larger than the value of 29024 calculated from the structure derived from the cDNA sequence (7). It should be noted here that a routine gel electrophoresis experiment would give a value of 30000 ± 3000, and consequently would not have picked up the difference observed with LDMS. To locate the source of the mass difference, the cyanogen bromide digest of S. aureus V8 protease was subjected to analysis by LDMS and fast atom bombardment mass spectrometry (FABMS). The molecular weights measured are

0097-6156/94/0549-O211$06.00/0 © 1994 American Chemical Society

In Time-of-Flight Mass Spectrometry; Cotter, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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TIME-OF-FLIGHT MASS S P E C T R O M E T R Y


listed in Table I and showed that the bulk of the molecular weight discrepancy was located in the C-terminal cyanogen bromide fragment. Treatment of the C-terminal cyanogen bromide fragment with α-chymotrypsin produced a peptide with 61 amino acids consisting almost entirely of proline, aspartic acid, and asparagine residues. Using mass spectrometry in conjunction with microchemical (Edman) sequencing, a sequence was derived that fits the requirements of the molecular weight data (4). The logic of this mass balance strategy requires that all pieces be accounted for at each step and that their sums correspond to the molecular weight determined originally. Analysis of the Tryptic Digest of a Glycoprotein Avidin, found in chicken eggs, is extensively glycosylated at a single asparagine site. LDMS was used to confirm the site and to determine the extent of glycosylation. After its disulfide bonds were reduced with dithiothreitol and the thiol groups alkylated with iodoacetamide, avidin was subjected to tryptic digestion. The products were divided in half. Half of the mixture was treated with water and half with an aqueous solution of N-glycosidase F, which catalyzes the cleavage of carbohydrate from asparagine side chains, leaving behind an aspartic acid residues. After partial fractionation by high performance liquid chromatography (HPLC), the various fractions from both the N-glycosidase F treated peptides and the non-treated peptides were analyzed by LDMS. Ions for most of the tryptic peptides predicted from the amino acid sequence (5) were found. Figure 1 shows the spectra obtained from fractions 6 of both treatments. The presence of a major peak may be noted in the Nglycosidase F treated fraction (panel a) that is absent in the non-treated fraction (panel b). This peptide has a molecular weight of 1836 daltons which corresponds to the tryptic peptide [10-26] whose asparagine at position 17 has been changed to an aspartic acid residue. Only micrograms of avidin were needed for the digestion, chromatography, and LDMS analysis. Laser desorption of the whole protein provided a broad signal, reflecting carbohydrate heterogeneity, with an average molecular weight of 16033 daltons. For this measurement, avidin was dissolved in 0.1 % trifluoroacetic acid (TFA) and then mixed with sinapinic acid (3, 5-dimethoxy-4-hydroxycinnamic acid) (6). The molecular weight of avidin was determined by sedimentation equilibrium in 1970, as 69300 (5), which indicates that 16033 is a measurement of the molecular weight of the subunit. TFA and sinapinic acid are undoubtedly denaturing agents. Subtracting the molecular weight of 14456 derived from the protein sequence provided the average weight of the carbohydrate, 1577 daltons. This indicates that avidin is about 10% carbohydrate by weight. The time required to obtain the LDMS spectrum of a tryptic digestfractionor of a protein is very short, on the order of two to five minutes using commercially available instrumentation. At least two or three days are needed to reduce and alkylate a protein, digest with trypsin, treat with N-glycosidase F, andfractionateby HPLC. This suggests that one laser desorption mass spectrometer can serve a number of biochemical researchers. Desorption of Proteins and Peptides from Membranes For LDMS to achieve its potential, it must be able to handle proteins and peptides as



12.

VESTLING

Laser Desorption Mass Spectrometry in Protein Analysi

Table I Molecular weight of the products of cyanogen bromide cleavage of 5. aureus V8 protease (a). peptide [1-117] [1-143] [118-143] [144-158] [159-end]

b

calc. M H 12520 15220 2701 1693 12020

+

observed m/z 12511 15243 2701 1693 13154

technique LDMS LDMS FABMS FABMS LDMS

*adopted from reference 3. Calculated from cDNA derived sequence, reference L


214

TIME-OF-FLIGHT MASS S P E C T R O M E T R Y

100 τ a)

[10-26] 1837

80 f

[72-87] 1896


60 f

[95-100]

404-

1819

U

204-

j3

Laser Desorption Mass Spectrometry in Protein Analysis - American

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