The determination of protein, oligonucleotide and peptide molecular weights by ion‐spray mass spectrometry

Covey, Thomas R.; Bonner, Ron; Shushan, Bori; Henion, Jack D.; Boyd, Robert K.

doi:10.1002/rcm.1290021111

Cited by 594 publications

(294 citation statements)

References 10 publications

Supporting

Mentioning

286

Contrasting

Unclassified

Order By: Relevance

“…Covey et al [27] found that the total number of basic residues (arginine, histidine, lysine, and the N-terminus) was often similar to the maximum charge state of a protein. Smith et al [4] also provided supporting details for this concept by compiling a list of proteins and peptides by including the maximum charge state obtained from ESI spectra and the number of basic sites.…”

Section: Methodsmentioning

confidence: 99%

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Wanasundara

Thachuk

2007

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

A series of calculations, varying from simple electrostatic to more detailed semi-empirical based molecular dynamics ones, were carried out on charged gas phase ions of the cytochrome c dimer. The energetics of differing charge states, charge partitionings, and charge configurations were examined in both the low and high charge regimes. As well, preliminary free energy calculations of dissociation barriers are presented. It is shown that one must always consider distributions of charge configurations, once protein relaxation effects are taken into account, and that no single configuration dominates. All these results also indicate that in the high charge limit, the dissociation of protein complex ions is governed by electrostatic repulsion from the net charges, the consequences of which are enumerated and discussed. There are two main trends deriving from this, namely that charges will move so as to approximately maintain constant surface charge density, and that the lowest barrier to dissociation is the one that produces fragment ions with equal charges. In particular, it is shown that the charge-to-mass ratio of a fragment ion is not the key physical parameter in predicting dissociation products. In fact, from the perspective of the division of total charge, many dissociation pathways reported to be "asymmetric" in the literature should be more properly labelled as "symmetric" or "near-symmetric". The Coulomb repulsion model assumes that the timescale for charge transfer is faster than that for protein structural changes, which in turn is faster than that for complex dissociation. One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass. Understanding the cause of this phenomenon will help to improve our understanding of the structural properties of noncovalent complexes.It is important to investigate how these dissociations occur and the factors that influence them. Using Fourier-transform mass spectrometry (FTMS), Jurchen and Williams [6] have conducted a number of studies in order to understand the origin of these charge distributions. In these experiments, isolated charge states of cytochrome c dimer were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD). According to Jurchen and Williams [6], the asymmetric charge distribution depends upon charge state, dissociation energy, and conformational flexibility. These studies showed that higher charge states lead to symmetric charge products while lower charge states lead to an asymmetric charge dissociation pathway. Further, their results with different excitation energies show ...

show abstract

Section: Methodsmentioning

confidence: 99%

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Wanasundara

Thachuk

2007

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…The average molecular mass of the glycopeptide (or peptide) can be calculated from the spectra based on the observed masses for more than one defined charge state of the molecule (Covey et al, 1988;Fenn et al, 1989). For example, Figure 3B reveals peaks at m/z 1,073.9 and m/z 1,610.1.…”

Section: Selective Detecfion Of Glycopeptidesmentioning

confidence: 99%

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

1993

View full text Add to dashboard Cite

A mass spectrometry method has been developed for selective detection of glycopeptides at the low (525) picomole level during chromatography of glycoprotein digests and for differentiation of 0-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in 4 2 , the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc'). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from 0-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase E These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three 0-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.

show abstract

“…Several factors have been shown to influence the observed charge state distribution, including molecular conformation [4][5][6], acid-base chemistry both in solution and in the gas phase [7][8][9][10][11], solvent composition [8], instrumental factors, etc. Several models have been proposed to qualitatively account for some of these effects [12][13][14][15]. A general conclusion from several studies is that the electrospray charge state distributions of proteins formed from denaturing solution conditions are shifted to higher charge states (lower m/z) than those formed from solutions in which the protein has significant tertiary structure.…”

Section: Introductionmentioning

confidence: 99%

Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization

Iavarone

Jurchen

Williams

2000

J. Am. Soc. Mass Spectrom.

172

199

View full text Add to dashboard Cite

The effects of solvent composition on both the maximum charge states and charge state distributions of analyte ions formed by electrospray ionization were investigated using a quadrupole mass spectrometer. The charge state distributions of cytochrome c and myoglobin, formed from 47%/50%/ 3% water/solvent/acetic acid solutions, shift to lower charge (higher m/z) when the 50% solvent fraction is changed from water to methanol, to acetonitrile, to isopropanol. This is also the order of increasing gas-phase basicities of these solvents, although other physical properties of these solvents may also play a role. The effect is relatively small for these solvents, possibly due to their limited concentration inside the electrospray interface. In contrast, the addition of even small amounts of diethylamine (<0.4%) results in dramatic shifts to lower charge, presumably due to preferential proton transfer from the higher charge state ions to diethylamine. These results clearly show that the maximum charge states and charge state distributions of ions formed by electrospray ionization are influenced by solvents that are more volatile than water. Addition of even small amounts of two solvents that are less volatile than water, ethylene glycol and 2-methoxyethanol, also results in preferential deprotonation of higher charge state ions of small peptides, but these solvents actually produce an enhancement in the higher charge state ions for both cytochrome c and myoglobin. For instruments that have capabilities that improve with lower m/z, this effect could be taken advantage of to improve the performance of an analysis.Electrospray ionization (ESI) [1] is well recognized as a soft ionization method for producing gas-phase ions of large biopolymers, such as oligonucleotides, proteins, and even noncovalent biomolecular complexes [2]. In combination with mass spectrometry, molecular masses of large molecules can be measured with unprecedented accuracy [3]. The multiple charging of large analyte ions that occurs with ESI has the advantage that the masses of very large molecules can be measured using mass spectrometers with upper mass-to-charge limits. One outstanding challenge in ESI mass spectrometry is to accurately predict the observed charge state distribution of a large molecule, given its primary structure, the composition of the solvent system from which the ions are formed, instrumental conditions, etc. Several factors have been shown to influence the observed charge state distribution, including molecular conformation [4][5][6], acid-base chemistry both in solution and in the gas phase [7][8][9][10][11], solvent composition [8], instrumental factors, etc. Several models have been proposed to qualitatively account for some of these effects [12][13][14][15]. A general conclusion from several studies is that the electrospray charge state distributions of proteins formed from denaturing solution conditions are shifted to higher charge states (lower m/z) than those formed from solutions in which the protein has signific...

show abstract

The determination of protein, oligonucleotide and peptide molecular weights by ion‐spray mass spectrometry

Cited by 594 publications

References 10 publications

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization

Contact Info

Product

Resources

About