Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%–15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients’ informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.

ICPL_ESIQuant is a proteomics software tool for quantitatively analyzing large mass spectrometric datasets acquired from ICPL based proteomics experiments. It is able to process mass spectrometric data from various vendors and implements results from the Mascot search engine to generate protein and peptide result tables. This protocol briefly introduces ICPL_ESIQuant and presents a detailed step by step tutorial, how to use the software with MS datasets obtained from ICPL duplex, triplex and quadruplex experiments. Requiring MS raw data in .mzXML file format and Mascot search results in .dat format as input, ICPL_ESIQuant reliably quantifies ICPL labeled proteins and provides additional information about all detected, sequenced and identified features in the sample. The software supports both the shotgun and the directed proteomics strategy, enabling the user to apply mass inclusion lists for identifying peptides not fragmented in the first MS cycle. The software together with a test dataset is freely available under http://sourceforge.net/projects/icplquant/. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Background: Known genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[ H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[ H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress.Methods: Protein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways.Results: The protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox- metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10 [ H213Q] subjects' and the ASD patients' set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress.Conclusions: Redox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox- sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.

: hemorphin, calcineurin, DJ-1 protein. ApolipoE, dopamine A growing body of data demonstrates that hemorphins, a family of endogenous nonclassical opioid peptides derived from hemoglobin (Hb) [22], exhibit a wide spectrum of biological activity by affecting different receptor’s function (e.g. µ-, δ- and к-opioid receptors [39], angiotensin (Ang) IV receptor (AT

By using the Isotope-Coded Protein Label (ICPL) approach [1,2] up to four different proteomics states can be compared with regard to their relative protein amounts. Measuring the samples using Liquid Chromatography coupled to Mass Spectrometry (LC-MS) results in hundreds and thousands of MS signals, each comprising m/z, intensity and retention time information of a certain compound ion. In order to detect and quantify peptides and proteins, many different software tools have been developed in the recent years, most of them comprising very intelligent peak picking and deisotoping algorithms [3].

In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis.

A great variety of technologies using stable isotope labeling in combination with mass spectrometry have been described being tools to identify and relatively quantify proteins within complex mixtures. Here, we present a method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on tagging stable isotope derivatives at the free amino groups of intact proteins, the method is applicable to any protein sample, including extracts from tissues or body fluids. All separation methods currently employed in proteome studies can be used to reduce complexity on the protein level. After enzymatic cleavage of the protein fractions, the ratios of peptides from different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides representing different expression levels in the different proteomic states are further analyzed by tandem-mass spectrometry to identify respective proteins. For quantification of proteins from multiplexed ICPL experiments, ICPLQuant was developed, a software package especially designed to cover the whole ICPL workflow. The ICPL method results in accurate and reproducible quantification of proteins and high sequence coverage, indispensable for a comprehensive detection of posttranslational modifications and discrimination of protein isoforms.

2‐DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off‐gel) of proteins with the help of a 12‐ or 24‐well frame. With this instrument also conventional focusing in IPG strips after passive in‐tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup‐loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in‐tray rehydration loading. Especially, the recovery of basic and high‐molecular‐mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48‐well OFFGEL frame was developed, which doubles the resolution of the commercially available 24‐well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.

Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle. The vast majority of presequences are proteolytically removed by the mitochondrial processing peptidase (MPP) localized in the matrix. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. However, the function of this second cleavage step is elusive. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins.

Stable isotope labeling in combination with mass spectrometry has emerged as a powerful tool to identify and quantify thousands of proteins within complex protein mixtures. Isotope-coded protein label (ICPL) is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the free amino groups of intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids. After labeling of up to four different proteome states, the samples can be combined and the complexity reduced by any separation method currently employed in protein chemistry. After enzymatic cleavage of the protein fractions the ratios of peptides in the different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides that exhibit regulations in the different proteome states are further investigated for identification by tandem-mass spectrometry. The quantification of multiplexed ICPL experiments is greatly facilitated by the recently published ICPLQuant software, which was especially designed to cover the whole ICPL workflow. The method shows highly accurate and reproducible quantification of proteins, yields high sequence coverage, and is indispensable for the comprehensive detection of posttranslational modifications and protein isoforms.

A rabbit antibody was produced against AL kappa(I) (UNK). It strongly reacted with amyloid on formalin-fixed paraffin embedded tissue sections of the same patient and detected AL kappa-amyloid of many other patients. In contrast, antibodies produced against kappa BJP of subclasses kappa(I)-kappa(IV) failed to label AL kappa (UNK) amyloid deposits.The patient continues to be free of systemic disease, already for 14 years until today.

Despite worldwide efforts biomarker discovery by plasma proteomics was not successful so far. Several reasons for this failure are obvious. Mainly, proteome diversity is remarkable between different individuals and is caused by genetic, environmental and life style parameters. To recognize disease related proteins that could serve as potential biomarkers is only feasible by investigating a non realizable large number of patients. Furthermore, plasma proteomics comprises enormous technical hurdles for quantitative analysis. High reproducibility of blood sampling in clinical routine is hard to achieve. Quantitative proteome analysis has to struggle with the complexity of millions of protein species comprising typical plasma proteins, cellular leakage proteins and antibodies and concentration differences of more than 1011 between high and low abundant proteins. Therefore, no successful quantitative and comprehensive plasma proteome analysis is reported so far. A novel proteomics strategy is proposed for biomarker discovery in plasma. Instead of comparing the plasma proteome of different individuals it is recommended to analyze the proteomes of different time points of a single individual during the development of a disease. This strategy is realized by the use of plasma of the Bavarian Red Cross Blood Bank, were three million samples are stored under standardized conditions. To achieve reliable data the isotope coded protein labelling proteomics technology was used.

The effect of synthetic LVV-hemorphin-7 and hemorphin-7 on hypothalamo-pituitary-adrenocortical axis activity in response to endotoxin-induced stress was studied. The intraperitoneal (ip) endotoxin (lipopolysaccaride, LPS) (0.5 mg/kg) administration in combination with hemorphin (1 mg/kg) induce significant decrease in plasma corticosterone and modest decrease in plasma levels of tumor necrosis factor-alpha (TNFalpha) in compare with elevated levels of both corticosterone and TNFalpha in plasma of rats received LPS administration alone. Increased activity of calcineurin in both plasma and brain of rats received ip administration of LPS, was recovered under LPS + hemorphin treatment. In two independent proteome analysis, using 2-dimensional fluorescence difference gel electrophoresis and the isotope coded protein label technology, peptidyl-prolyl cis-trans-isomerase A (cyclophilin A) was identified as regulated by hemorphins protein in mouse brain. A therapeutic potential of hemorphins and mechanisms of their homeostatic action in response to endotoxin-induced stress are discussed.