4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III. Proteomes and Post-Translational Modifications 285 A. Proteomes 285 1. The Analytical Challenge 285 2. Analysis of Protein−Protein Complexes 286 B. Post-Translational Modifications 286 1. Background 286 2. Detection and Purification of Phosphoproteins 288

Mass Spectrometry in Proteomics

Electrospray ionization mass spectrometry has been used to study protein interactions driven by noncovalent forces The gentleness of the electrospray ionization process allows intact protein complexes to be directly detected by mass spectrometry Evidence from the growing body of literature suggests that the ESI-MS observations for these weakly bound systems reflect, to some extent, the nature of the interaction found in the condensed phase Stoichiometry of the complex can be easily obtained from the resulting mass spectrum because the molecular weight of the complex is directly measured For the study of protein interactions, ESI-MS is complementary to other biophysical methods, such as NMR and analytical ultracentrifugation However, mass spectrometry offers advantages in speed and sensitivity The experimental variables that play a role in the outcome of ESI-MS studies of noncovalently bound complexes are reviewed Several applications of ESI-MS are discussed, including protein interactions with metal ions and nucleic acids and subunit protein structures (quaternary structure)

Studying noncovalent protein complexes by electrospray ionization mass spectrometry

There is an advantage for users of electrospray and nanospray mass spectrometry to have an understanding of the processes involved in the conversion of the ions present in the solution to ions in the gas phase. The following processes are considered: Creation of charge droplets at the capillary tip; Electrical potentials required and possibility of gas discharges; Evolution of charged droplets, due to solvent evaporation and Coulomb explosions, to very small droplets that are the precursors of the gas phase ions; Production of gas phase ions from these droplets via the Ion Evaporation and Charge residue models; Analytical uses of ESIMS of small ions, qualitative and quantitative analysis; Effects of the ESI mechanism on the analysis of proteins and protein complexes; Determination of stability constants of protein complexes; Role of additives such as ammonium acetate on the observed mass spectra.

Electrospray: From ions in solution to ions in the gas phase, what we know now

A brief account of the mechanisms by which ions in solution are converted to ions in the gas phase is given on the basis of information available in the literature and the four companion articles on electrospray mass spectrometry (ESMS) in this issue. The following stages/phenomena are described: (a) production of the charged droplets at the ES capillary tip; (b) evolution of the charged droplets due to solvent evaporation and droplet fission caused by Coulombic repulsion of the charges on the droplets; production of the gas phase ion from very small charged droplets by the charge residue model (CRM) or the ion evaporation method (IEM); (c) dependence of the sensitivity in ESMS on the chemical nature of the analyte and its concentration as well as on the concentration of other electrolytes that are present in the solution; qualitative predictions on the sensitivity of the analyte based on the surface activity of the analyte ions; (d) relationship between ions produced in the gas phase and original ions present in the solution; and (e) globular proteins. Much of the information presented in (a)-(e) has been available for some time in the literature. However some significant advances are relatively recent. Recent results by de la Mora and co-workers, including their contribution in this Special Feature, provide very strong evidence that small ions (in distinction from macroions such as bio-macroions) are produced by IEM. On the other hand, macroions and particularly the polyprotonated globular proteins are produced by CRM. Also noteworthy is the development of an equation by Enke with which the observed relative ion signal intensities of the gas-phase ions produced can be predicted on the basis of the ion concentration in solution over a wide concentration range. The recognition that the sensitivity of organic analyte ions can be qualitatively predicted on the basis of the hydrophilicity or hydrophobicity of the part of the molecule that is not part of the charged (ionic) group and affects the surface activity of the ionic species is also noteworthy and a very useful relatively recent development.

A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry

Principles and practice of electrospray ionization—mass spectrometry for large polypeptides and proteins

Detection of noncovalent receptor-ligand complexes by mass spectrometry

Observation of noncovalent enzyme-substrate and enzyme-product complexes by ion-spray mass spectrometry

Detection of oligonucleotide duplex forms by ion-spray mass spectrometry

The leucine zipper, found in several DNA-binding proteins, represents a motif for noncovalent dimerization to enhance DNA binding. Solution dimerization of the leucine zipper peptides GCN4-p1 and N16V is confirmed here by on-line, size-exclusion liquid chromatography-ion spray mass spectrometry. Tandem mass spectrometry studies indicate that dimer ions also exist in the gas phase. High-resolution trapped-ion studies show that these gaseous dimers are stable for at least minutes. In qualitative agreement with their behavior in aqueous solution, N16V has a greater tendency to form dimers than does GCN4-p1, which unexpectedly forms a noncovalent heterodimer with an impurity

Mass spectrometric studies on noncovalent dimers of leucine zipper peptides

The synthesis of bacteriophage T4 DNA is catalyzed by a complex of five proteins that includes the T4 DNA polymerase protein, one single-stranded DNA binding protein, and three T4-coded polymerase accessory proteins. The accessory proteins form two subassemblies, one consisting of a tightly-bound complex of gene 44 and 62 proteins and the other an oligomer of the gene 45 protein (product of gene 45, gp45). On the basis of equilibrium sedimentation, velocity sedimentation, and chemical cross-linking studies, aqueous gp45 was thought to exist as a noncovalent trimer at pH 7. However, independent studies employing a combination of gel filtration and sucrose gradient sedimentation suggested that gp45 exists as a dimer in solution

Yu Tsyr Li

Papers

Detection of noncovalent receptor-ligand complexes by mass spectrometry

Observation of noncovalent enzyme-substrate and enzyme-product complexes by ion-spray mass spectrometry

Detection of oligonucleotide duplex forms by ion-spray mass spectrometry

Mass spectrometric studies on noncovalent dimers of leucine zipper peptides

Analysis of the Stoichiometry of the T4 Gene 45 Protein by Ion Spray Mass Spectrometry