Current Proteomics - Volume 6, Issue 1, 2009

The analysis of antibodies in human serum is an established technique in the laboratory diagnosis of infectious as well as autoimmune diseases. The multitude of antibody reactions towards pathogens and likewise the antibody profile in autoimmune diseases does contain a wealth of proteomic (antibody) data that may constitute valuable diagnostic information with relevance for the patient's prognosis and response to therapy. Hence the use of antibodies as diagnostic biomarkers may be one of the most promising strategies to identify patient subgroups. The presence or absence of antibodies directed against specific epitopes could represent a serologic biomarker that is able to predict the severity of a disease and assist in medical decision making. In addition, parallel detection of many different antibodies in a serum sample would be of great value in many areas of basic immunological research. Peptide arrays displaying biologically active small synthetic peptides in either low, medium or high-density formats represent an attractive technology to probe complex serum samples for the presence of such antibody analytes. Holding the unique capacity to break down the heterogeneous immunologic response into monoclonal antibody specificities and to differentiate subtle changes in antibody abundance and specificity, the peptide array technology by far extends the diagnostic potential of any conventional serologic assay. Together with an unrivalled parallelity, peptide (micro)array analysis opens new perspectives for the novel use of antibodies as diagnostic biomarkers and provides unique access to a more differentiated serological diagnosis. This review recapitulates the development of the peptide array technology with a focus on recent advances and current state of the art platforms for antibody diagnostics. Latest applications of peptide arrays for the serologic diagnosis of infectious diseases, autoimmunity and allergy are discussed, and conclusions for future developments and implications are drawn.

The adaptive immune response relies on the ability of T-lymphocytes to recognize small antigenic peptides presented on the cell surface by specialized receptors of the Major Histocompatibility Complex (MHC). These peptides are either generated by the degradation of intracellular proteins (MHC class I pathway) or by the degradation of internalized extracellular proteins (MHC class II pathway and cross-presentation pathway). The number of proteins that can be degraded by these pathways runs to the thousands leading to a staggering number of possible peptide fragments. A small subset of these peptides is selected by the cell's processing and presentation mechanisms to be presented on the cell surface by MHC molecules and has been defined as the immunopeptidome. The peptide sequences that comprise the immunopeptidome control the immune response and variations of this peptide repertoire are key to understanding the host's ability to fight pathogens, immune response to cancer as well as predisposition to autoimmunity and allergies. In the last few years it has been established that the composition of the immunopeptidome is regulated by specific cellular mechanisms that influence qualitative and quantitative aspects of the cellular immune response in a process that has been described as antigenic peptide editing. This review explores the current knowledge on these cellular mechanisms and discusses the parallels between editing the MHC class I and class II immunopeptidomes.

The versatile affinity purification techniques for isolating mammalian protein complex in combination with mass spectrometry-based proteomics are powerful to decipher the characters of the associated binding partners within a protein complex and protein-protein interactions. One single epitope-tag affinity purification for purifying protein complex is variable and limited in protein purity and specificity for a different individual bait protein. Recently, several newly developed tandem affinity purification (TAP) systems have been applied to isolate the native protein complexes with high purity and specificity at close to physiological levels in mammalian cells, furthermore a novel quantitative MAP (mixing after purification)-SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometric technique integrated with affinity purification effectively investigates weak associated partners as well as deciphers specific and dynamic protein-protein interactions.

Phosphorylation is one of the most important and ubiquitous modifications in eukaryotic cells. This covalent modification is a major signaling pathway in living beings. A vast array of cellular events, such as proliferation, differentiation, metabolism, signal transduction, and adaptation to environmental stress, and the function of many proteins, hormones, neurotransmitters, and enzymes, are triggered by phosphorylation. For understanding highly interconnected regulatory network, it is essential to identify and quantify phosphoproteins in biological specimens. Currently, this task is accomplished by mass spectrometry-driven phosphoproteomics. This article outlines recent developments in the analysis of phosphoproteins, specifically, the enrichment, detection, identification, and quantification of phosphopeptides/phosphoproteins.

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers, we applied OFFGEL-IEF, iTRAQ labeling and mass spectrometry (LC- MS/MS) analysis to identify proteins in MDA-MB-435 breast cancer cell line. Protein expression profiling might yield valuable insights into molecular signals which play important role in metastatic progression. Approximately 1250 proteins were identified with >95% confidence. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1, annexin I and and tumor protein D54 were identified as important cellular proteins. Most of these proteins that were identified are involved in cell growth, metabolism, signal transduction and transcriptional activation. An integrated approach for mining and visualization of iTRAQ data is presented. The results provide an initial assessment of the proteome in MDA-MB-435 breast cancer cell line, and an improved understanding of metastasis tumor progression.

Identification of thiol modifications has gained significant importance. It is increasingly recognized that cysteines play an important role in protein function under both physiological and patho-physiological conditions. Here we reviewed different approaches that are used to identify oxidized proteins and discuss different fluorescent labeling techniques, differential two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization - time of flight identification, in short MALDI-TOF. We illuminate processes that depend on protein oxidation of cysteines and we look into consequences of thiol oxidation during aging and in a variety of diseases, with a special reference to Alzheimer's disease. There is an urgent need for methods that detect specifically oxidized proteins and are able to distinguish different oxidation types.

The vast majority of clinical tissue samples are formalin-fixed and paraffin-preserved. This type of preservation has been considered an obstacle to protein extraction from these tissues. However, these are the very tissue samples that have associated patient histories, diagnoses and outcomes - ideal samples in the quest to translate bench research into clinical applications. Thus, until recently, these valuable specimens have been unavailable for proteomic analysis. Over the last decade, researchers have been exploring efficient methods to undo protein cross-linking caused by standard tissue fixatives and extract proteins from archived tissue specimens. These methods have been applied in different clinical proteomic studies. In this report, we attempt to review the development of these techniques, summarize the proteomic findings, and discuss the impact on future clinical proteomics.