Current Proteomics - Volume 7, Issue 3, 2010

Protein post-translational modifications (PTMs) play a critical role in modulating cellular functions. PTMs also vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are sub-stoichiometric, transient and reversible. However, accurate qualitative and quantitative mapping of modification sites on proteins is of immense value because it may provide specific biomarkers and protein targets for various biological and physiological conditions. In recent years, a wide variety of proteomic approaches using gel electrophoresis, liquid chromatography and mass spectrometry has been developed to detect PTMs, and many exciting results produced by using sophisticated procedures integrating innovative separation and enrichment methods along with advanced mass spectrometric analyses have been published. This special issue aims to summarize recent progresses in PTM proteomics and posts further directions for the biomarker and target identification by proteomics.

Glycoproteomic researches have been rapidly progressing in the last few years. Although intact glycopeptide analysis which obtains glycosite and corresponding glycan information simultaneously is still challenging, various studies, combining glycosylation enrichment methods and mass spectrometry techniques, have accumulated a considerable amount of protein glycosite data for either N- or O-glycosylation or O-GlcNAcylation. This review will majorly focus on recent research outputs in the field of glycosite exploration.

Protein phosphorylation plays a key role in the regulation of most cellular processes and the disorder of this process often leads to illness or even cancers. Panoramic phosphorylation analysis of organisms known as “phosphoproteomics” has become more important and popular in scientific research fields these days due to its ability of gaining significant information about phosphorylation events and its assistance in understanding how those regulatory functions work in cell. Analytical strategies have been developed for comprehensive analysis of phosphoproteome over the past several years on the aspects of both profiling and quantification. In this review, we will discuss some of the recent applications of phosphoproteomics by using several commonly used technologies, and the applications on study of cell signal transduction pathways and disease-related biomarker and drug targets discovery will also be covered.

Biological research in recent years has been expedited by the advent of mass spectrometry-based proteomics. In the area of chromatin research, mass spectrometry has emerged as an important platform for characterizing the specific locations and types of post-translational modifications (PTMs) present on histone proteins. Substantial evidence supports the claim that chromatin structure is modulated by the “Histone Code” hypothesis, which states that specific posttranslational modifications (PTMs) of the histone proteins on the nucleosomes drive the formation of euchromatin (i.e., open and transcriptionally active chromatin) and heterochromatin (i.e., compact and transcriptionally repressed chromatin) by signaling and interacting with the appropriate transcriptional and chromatin remodeling machinery. Thus, the ability to accurately discover, identify, and quantify these modified forms is of immense value to the field. This article reviews the various mass spectrometric methods that have recently been developed for the study of histone variants and their posttranslational modifications.

Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial enzymes that are unique in exploiting the ATP/GTPbinding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic detection of phosphorylated tyrosines by phosphoprotomics studies in bacteria. Other pioneering studies conducted in recent years, such as the first structures of BY-kinases and biochemical and phyiological studies of new BY-kinase substrates significantly furthered our understanding of these enzymes and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design.

Application of serum proteomics and peptidomics to the field of haematological malignancies can lead to an in depth understanding of the evolution of leukemogenesis, offering potential for early diagnosis, individualized and molecularly specific treatments, and disease surveillance through the monitoring of protein signatures. Currently, the limited understanding of aetiology prevents curative treatments for haematological malignancies. Until recently, the primary focus was on cytogenetics. Gene expression profiling and metaphase karyotyping has been done extensively, yet the lack of correlation between transcript and protein has limited the comprehensiveness of these approaches. Transcriptomics has dominated the field with significant emphasis being placed upon SNP genotyping, copy number variation analysis, DNA sequencing and methylation profiling. However, the integration of the existing knowledge with proteomics is needed to discover the molecular mechanisms of haematological malignancies and for the development of diagnostic tests. Proteomic analysis allows for direct assessment of the expressed, modified and degraded proteins. The Human Serum and Plasma Proteome Projects coupled with advancing technologies with increased sensitivity and throughput are beginning to produce productive breakthroughs including new markers for the detection of early stages of leukaemias and lymphomas. This review summarizes recent research efforts in the field of haematological malignancies, and examines relevant high throughput proteomic technologies, limitations, and pathophysiological background. A broader perspective on candidates for marker panels for the detection of malignant transformations is discussed.

The present-day genetic code, while universal, is not the result of a “frozen accident”, but has evolved over time. Analysis of amino-acid frequency has recently identified amino acids that were recruited early and late into the genetic code. We use this observation to lend support to a hypothesis that the current quaternary triplet genetic code comes from an earlier quaternary doublet code, and before that from a binary singlet code.

Colorectal cancer is one of the most common cancers and has one of the highest cancer mortality rates. New therapies are needed in order to specifically target individual patients to reduce toxicities and improve patient outcome in the clinic. The potential for biomarker discovery to impact current understanding and outcome of colorectal cancer has yet to be fully achieved, with many potential biomarkers identified but lack of sensitivity and/or specificity in the clinical setting render them inadequate. Biomarkers can provide prognostic or predictive information, act as screening tools or therapeutic targets and improve patient survival, yet thus far their use has been elusive in colorectal cancer. Proteomic techniques are one of the most widely used tools to enable biomarker detection due to its ability to assess protein expression, activation, post-translational modifications as well as its high-throughput potential, robustness and accuracy. This review focuses on the use of proteomic techniques for the identification of potential biomarkers and the use of biomarkers in improving patient therapies.

Intracellular bacterial pathogens remain a major cause of infection leading to human diseases and death worldwide. With the advent of proteomic technologies, a number of proteomic studies of intracellular bacteria have been carried out to gain insight into the adaptation, survival, or proliferation of bacteria inside host cells. Over 20 intracellular bacterial species have been investigated in various host cells. In this review, we summarize the intracellular bacteria studied by mass spectrometry- or immunostaining-based proteomic techniques, the number of proteins identified for the intracellular bacteria, and the implications of the results in the interaction between the bacteria and their hosts. We also discuss the factors that limit the depth of current proteomic studies of intracellular bacteria and the possible approaches to overcome the limitations.