Little more than three years down the line and I am already writing the Preface to a second volume to follow Protein and Peptide Analysis by Mass . What has happened in between these times to make this second venture worthwhile? New types of mass spectrometric instrumentation have appeared so that new techniques have become possible and existing techniques have become much more feasible. More particularly, however, the newer ionization te- niques, introduced for the analysis of high molecular weight materials, have now been thoroughly used and studied. As a result, there has been an en- mous improvement in the associated sample handling technology so that these methods are now routinely applied to much smaller sample amounts as well as to more intractable samples. Again, this particular community of mass spectrometry users has both increased in number and diversified. And, riding this wave of acceptance, leaders in the field have set their sights on more complex problems: molecular interaction, ion structures, quantitation, and kinetics are just a few of the newer areas reported in Mass Spectrometry of Proteins and Peptides. As with the first volume, one purpose of this collection, Mass Spectr- etry of Proteins and Peptides, is to show the reader what can be done by the application of mass spectrometry, and perhaps even to encourage the reader to venture down new paths.

In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg

Oxidative stress is recognized as an important underlying factor in the pathogenesis of many degenerative diseases as well as normal senescence. The free radicals, reactive oxygen species (ROS) and electrophiles produced during oxidative stress are capable of modifying nucleic acids, lipids and proteins. There are a variety of oxidative modifications that occur to proteins including: cleavage of the protein backbone, direct oxidation of amino acid side chains by ROS, and adduction by electrophilic species such as lipid peroxidation products. Many of these oxidative modifications result in the introduction of carbonyl groups into the proteins. Protein carbonylation levels are commonly used as a biomarker to assess the degree of oxidative damage to a system. However the most commonly employed methods for measuring oxidative modifications to proteins, typically fail to provide any information about the identity of the modified protein, site of modification, or the chemical nature of the modification. In the present study we develop an analytical technique based on affinity labeling with N'-aminooxymethylcarbonylhydrazino-D-biotin (aldehyde reactive probe, ARP), along with mass spectrometric analysis which allows for the full characterization of protein carbonylation modifications. The ability of the ARP method was first demonstrated for the case of oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions with [alpha,beta]- unsaturated aldehydic lipid peroxidation products with nucleophilic amino acid residue side chains. ARP was used to label a 4-hydroxy-2-nonenal (HNE) modified cysteine containing model peptide, and HNE modified E. coli thioredoxin, which were characterized using ESI-MS/MS and MALDI-MS/MS. ARP was also used to label the oxidative modifications alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS), formed during the metal catalyzed oxidation of GAPDH. After demonstrating the utility of the technique on model systems, it was then applied to complex biological systems. In one case, subsarcolemmal mitochondria (SSM) isolated from rat cardiac tissue. Mitochondria are well known to be a major source of ROS within the cell. They are therefore important mediators of oxidative stress, as well as regulators of cell death. We were able to identify 39 unique sites on 27 mitochondrial proteins which were modified by six different [alpha,beta]-unsaturated aldehydes, including acrolein, [beta]-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2- nonenal. Additionally we identified nine Lys residues on four mitochondrial proteins that were oxidized to AAS and subsequently labeled with ARP. The proteins identified with oxidative modifications include members of the mitochondrial electron transport chain, TCA cycle, membrane transport, lipid metabolism, and other important mitochondrial enzymes. The ARP technique was also applied to identify protein targets of 4-hyroxy-2- nonenal in human monocytic THP-1 cells that were exogenously exposed to HNE. It was shown previously that exposure of THP-1 cells to HNE resulted in apoptosis, necrosis and protein carbonylation. We applied a multi-pronged proteomic approach involving electrophoretic, immunoblotting and mass spectrometric analysis to unequivocally identify eighteen sites of HNE modification on sixteen proteins. It was also demonstrated in this study that pretreatment of THP-1 cells with ascorbic acid resulted in decreased levels of HNE-protein conjugate formation.

Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

At present where protein identification and characterisation using mass spectrometry is a method of choice, this book is presenting a review of basic proteomic techniques. The second part of the book is related to the novel high throughput protein identification technique called the 'molecular scanner'. Several protein identification techniques are described, especially the peptide mass fingerprint with MALDI-MS based method. E.g. ionisation process, matrix available, signal reproducibility and suppression effect, as well as date treatment for protein identification using bioinformatics tools.