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

Interaction of membrane proteins and lipids with solubilizing detergents.

Ion formation in MALDI: the cluster ionization mechanism.

A review on the application of mass spectrometry (MS) to the upper levels of protein organization is presented A description on the processes of generating, transmitting, and analyzing ions formed by protein complexes is presented as well as the experimental conditions theory that are the standards for the investigations In addition, some other approaches are included that can be used in conjunction with MS of intact complexes This review aims to establish MS of intact complexes as part of future structural genomic and proteomic investigations Several other topics discussed include the generation of ions of protein complexes, the transmission and analysis of ions o protein complexes, the activation and dissociation of protein complexes, and lastly, the companion technologies for MS of protein complexes

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Protein complexes in the gas phase: technology for structural genomics and proteomics.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for mass fingerprinting of peptide mixtures obtained after enzymatic in-gel digestion of proteins separated by two-dimensional electrophoresis (2-DE). In the course of a proteome analysis of mycobacteria using mass spectrometric identification, it was found that 94% of the most intense MALDI-MS peaks denote peptides bearing arginine at the C-terminal end. The effect was demonstrated to be equally prominent using an equimolar mixture of the synthetic peptides known to be present in the tryptic digest of the mycobacterial 35 kDa antigen (“synthetic mass map”). In addition, several binary mixtures of synthetic peptides differing exclusively at the C terminus (Arg or Lys) were examined to rationalize the higher sensitivity toward arginine-containing peptides. The extent of the effect described depends on the matrix used and may facilitate a more reliable assignment of mass fingerprint data to protein sequences in datab...

The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins.

Matrix-assisted laser desorption/ionization (MALDI) mass spectra and methods to improve their quality are reported for three hydrophobic, membrane-bound proteins: porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium and cholesterolesterase from Pseudomonas fluorescens. Several commonly used UV and IR matrices have been tested. In addition, the susceptibility of MALDI mass spectrometry to various neutral and ionic detergents, known usually to degrade the quality of MALDI mass spectra, has been tested systematically. For porin, consisting of three identical non-covalently bound subunits, a new sample preparation is reported, resulting in the desorption of the intact quaternary protein structure. This leads to a better understanding of the way a given analyte is embedded into the host matrix crystals.

Ulrike Krüger

Papers

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of membrane proteins and non-covalent complexes