Complications in the Determination of Molecular Weights of Proteins

Chapter 22

Downloaded by STANFORD UNIV GREEN LIBR on October 11, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch022

Complications in the Determination of Molecular Weights of Proteins and Peptides Using Electrospray Ionization Mass Spectrometry 1

Catherine Fenselau and Michele Kelly

Structural Biochemistry Center, University of Maryland—Baltimore County, 5401 Wilkens Avenue, Baltimore, MD 21228 Complications are discussed that have been encountered in assigning molecular weights to proteins and peptides from electrospray mass spectra. A variety of causes can contribute molecular ions to spectra that are heavier or lighter than the actual values, or multiple peaks in the region of the molecular ion. Examples are taken from studies of the mutation and processing of gag preproteins inlentevirus,and from the determination of the primary structure of the orphan drug bovine adenosine deaminase. The demonstration of the ability of electrospray mass spectrometry to provide accurate molecular weights of proteins, its easy retrofit on mass spectrometers already in the field, and its compatibility as an interface between mass spectrometry and high pressure liquid chromatography or capillary electrophoresis have led to immediate acceptance and widespread implementation in mass spectrometry laboratories, and also in steadily increasing numbers of biochemistry laboratories. The application of mass spectrometry to biochemical problems has also been catalyzed by matrix assisted laser desorption ionization (MALDI). The reliable determination of molecular weights by electrospray is being exploited in protein folding studies that employ H/D exchange, determination of molar ratios in non-covalent complexes, and characterization of unstable reaction intermediates. Electrospray ionization mass spectrometry has also found widespread application in studies of protein processing and modifications of proteins by enzymic and chemical reactions. In the present report we discuss the reliability of molecular weight determinations made by electrospray with illustrations from studies of protein processing, post-translational modification, and peptide mapping. 1

Current address: Ciba-Geigy Corporation, 556 Morris Avenue, Summit, NJ 07901 0097-6156/95/0619-0424$12.00/0 © 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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As has been pointed out (1), molecular weight determinations by electrospray and MALDI mass spectrometry are significantly more accurate than estimations based on the widely used polyacrylamide gel electrophoretic (PAGE) methods. More significant figures are provided, and the measurement is not altered by hydrophobicity or chemical modification. Mass resolution (the ability to separate proteins with similar molecular weights) is also higher with mass spectrometry. Some examples of comparative detenninations are provided in Table I. Nonetheless, gel electrophoresis is a convenient and popular technique for proteins, and developmental efforts are underway to interface gel electrophoresis with MALDI and electrospray mass spectrometry.

Table I. Examples of Protein Molecular Weights Determined by Gel Electrophoresis and Electrospray Mass Spectrometry Protein Kinase C Nucleocapsid alpha amylase-1

PAGE 7000 23000 13000 45000

ESMS 10460 + 1,10537 17288 + 7, 17362 7283 ± 5 45529 + 8,45774 45833 + 9, 46077

+ 1 + 7 + 11 + 11

Reference 2 2 3 4

The general experience of the community also indicates that protein analysis by mass spectrometry provides information that is highly complementary to that inferred from gene sequences. Post-translational modifications, including processing, may be revealed by the lack of agreement between measured and inferred molecular weights, and occasionally the gene sequence is shown to be incorrect. Several examples are summarized in Table II. Although analysis by mass spectrometry does destroy the sample, its accuracy, sensitivity and speed make it the method of choice for direct examination of proteins.

Table Π. Examples of Protein Molecular Weights Determined by Gene Sequencing and Mass Spectrometry Protein Glyoxalasel S. aureus V8

Gene Sequence Mass Spectrometry Reference 18442 19440 + 16 5 29024 29994 ± 3 6

We have recently published collaborative studies of the processing of preproteins transcribed by the gag (group antigen) genes in both human immunodeficiency virus (HTV) (7) and bovine immunodeficiency virus (BIV) (3). In both cases the gene sequence had been determined, and molecular weights were expected to reveal proteolytic sites in the preprotein. Not unexpectedly, this straightforward strategy was compromised by the presence


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of modifications such as myristoylation of the amino terminus in HIV (though not in BIV), errors in the protein sequences deduced from gene sequences, and mutations that occurred during cell culture. Some of the results of these processing studies are summarized in Table ΙΠ. Assignments in the first and fourth columns are based on mobility in gel electrophoresis, and calculated values are based on gene and protein sequences. TABLE m. Electrospray Mass Spectrometry Analysis of Lentevirus Proteins Processed from gag Preproteins MW MW HIV (7) pl7 p24 p7

Calc'd 15056 25551 6451

Observed BIV (3) 15269 + 4 pl6 25752 + 50 p26 6451 ± 1 pl3

Calc'd 14627 24596 7287

Observed 14628 + 6 24610 + 14 7283 + 5

Observed values in Table ΠΙ were determined by electrospray mass spectrometry. Functions in cell growth and replication for the proteins in Table III have been assigned by others. Mutations were found to be associated frequently with pl7 from HTV, and to a lesser extent with p24 from HTV. However, no mutations were detected in p7 from HTV cells that had successfully replicated in culture. This is consistent with the requirement of this zinc finger protein for viral genome recognition during budding, genomic RNA packaging, and early events in viral infection. Several experimental problems were encountered in these molecular weight determinations that led to the detection of multiple molecular ions in some samples. After the cultured cells were lysed under appropriate biosafety conditions (7), proteins were stabilized in solutions of thioethanol and guanidine hydrochloride and fractionated by complex reverse phase high pressure liquid chromatography (HPLC). Even with rechromatography, complete purification was sometimes difficult and samples collected for mass spectrometry sometimes contained more than one protein (3). Considerable heterogeneity or genetic mutation was encountered, and the resulting protein and peptide isoforms often had very similar chromatographic behavior, but different molecular weights. One protein was converted to a mixture by partial phosphorylation in the cultured virus. The electrospray spectrum of gag p24 isolate from cultured HIV cells (8) was much more complex than that of HTV p24 prepared by recombinant techniques (9). Earlier work with capsid proteins from other HTV strains had revealed electrophoretic heterogeneity and partial phosphorylation (7). This is assumed to contribute to the complexity of the electrospray spectrum, along with sequence heterogeneity confirmed by Edman chemistry and collisional activation in tandem mass spectrometry experiments. In contrast, the molecular weight of the major form of p26 (>90%) isolated from BIV and determined by electrospray ionization, agreed to within 2 Da with that predicted by the gene sequence and was judged not to be phosphorylated.


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Multiple proteins can also derive from imprecise processing that leaves ragged protein ends. In this case the mass differences within a series of mass spectral peaks will correspond to additional amino acids at the amino or carboxyl termini. Ragged ends and thus mixtures can also be formed artifactually by chemical cleavages that occur during protein purification and preparation of samples for mass spectrometry. Mass spectrometry is poorly equipped to distinguish these histories. In theory, the protein could be purified in H O, under which conditions isotopically labelled oxygen would be incorporated into any artifactual hydrolysis products for recognition by mass spectrometry (10). Artifactual chemical modifications sometimes occur during protein purification and preparation of peptide maps. For example, thioethanol, frequently used in purification, can form disulfide bonds with unprotected cysteines, and sulfur in methionine is readily oxidized to sulfoxides and sulfones. Another source of multiple ions in mass spectra of proteins is cleavage in the electrospray ionization source of small pieces from either the amino or carboxyl terminus. This phenomena is generally attributed to collisions occurring in the electrospray source. It is usually manifest as a series of peaks whose mass differences correspond to amino acid residues, and it has been termed microsequencing by Tom Covey (11). A selection of examples is presented in Table IV, and includes cleavages at proline, glutamate and other residues. Primary structures and actual molecular weights of the proteins were established independently in the appropriate references. In at least one case (15) the denatured protein has been found to fragment more readily than the folded protein.


2

Table IV. Examples of Protein Truncation in the Electrospray Source Mol. Wt. Observed Neutral fragment 25554 acetyl 25469 acetyl-S 21978 M-F 15416 M 7122 A-S 5953 acetyl-M 5824 acetyl-M-D 3722 acetyl-A

Ionized protein S-Κ··· Κ ρ ·.·.·. A ··· Q ··· D-Ρ··· ρ ... Q

···

Reference 12 12 13 13 3 15 15 14

The amino acid and acetyl groups listed as neutral fragments were shown by independent means to be bonded to the amino acids shown in the next column as amino termini of the ionized proteins actually detected. The truncated proteins listed in Table IV were observed as fully protonated y class ions (9,16) formed by cleavages between the carbonyl and amino moieties of amide bonds near the amino terminus. It is difficult to make the distinction


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Complications in the Determination of Molecular Weights of Proteins

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