Karin M Hansson

Analysis of Gas6 in Human Platelets and Plasma

Objective—Gas6 is a member of the vitamin K-dependent protein family. Gas6-deficient mice were found to be resistant to thrombosis because of defective platelet function. Mouse Gas6 was demonstrated to be present in platelets and found to be involved in platelet aggregation. The aim of this study was to investigate the presence of Gas6 in human platelets and plasma and determine its role in platelet function. Methods and Results—The presence of Gas6 in human platelets and plasma was analyzed using sensitive immunologic methods. Mass spectrometry and ELISA were used to identify and quantify Gas6 in plasma. Gas6 was demonstrated to be present in human plasma, at a concentration determined to be 13 to 23 ng/mL (0.16 to 0.28 nM). Furthermore, plasma Gas6 levels were found to be lower in patients administered with warfarin. However, Gas6 was undetectable in human platelets. Conclusions—This is the first report to identify and quantify Gas6 in human plasma. However, Gas6 protein was not detected in human platelets, suggesting that any potential platelet-specific function could be because of Gas6 from the circulation. These findings open up new directions regarding the role of Gas6 in normal and pathophysiological situations such as inflammation, autoimmune disease, thrombosis and arteriosclerosis.

Post-translational modifications in proteins involved in blood coagulation.

Blood coagulation and its anticoagulant counterpart, the protein C (PC) system, proceed via the formation of cellassociatedmacromolecular enzyme complexes that interact in a precisely regulated manner. In the final step of coagulation thrombin is generated, which cleaves soluble fibrinogen to form the insoluble fibrin monomers that constitute the structural foundation of a blood clot [1,2]. Post-translational protein modifications play pivotal roles in both blood coagulation and the PC anticoagulant system. The relatively high concentration of most of the blood coagulation proteins enabled many of them to be purified to homogeneity some 30–40 or more years ago. Advanced chemical characterization and protein sequencing became possible, which in time resulted in the identification of several post-translational amino acid modifications. These developments, and the interest in blood coagulation and coagulation disorders, led to the identification of three types of modified amino acids in coagulation proteins before they were found in proteins belonging to other systems. The first was c-carboxyglutamic acid (Gla) in 1974, which is formed by vitamin K-dependent c-carboxylation of glutamic acid residues [3–5]. Then followed erythro-b-hydroxyaspartic acid (Hya) and erythro-b-hydroxyasparagine (Hyn), which are formed by hydroxylation of aspartic acid and asparagine residues, respectively [6,7]. At that time, blood coagulation was described by the so-called cascade scheme [2,8,9]. It called attention to the signal amplification that is required to obtain the amounts of thrombin necessary to convert soluble fibrinogen to insoluble fibrin and it illustrated the role of enzymatically active macromolecular complexes. Moreover, the importance of binding to cell surfaces, i.e. that the coagulation process to a large extent is an example of chemistry in two (rather than three) dimensions was only beginning to emerge. The identification of Gla, stimulated by a desire to learn about the mode of action of warfarin [10], indirectly led to the purification of PC, and a few years later protein S and thrombomodulin [11–15]. With these proteins identified, and with the novel co-factor role of factor V established as a result of research on resistance to activated protein C (APC), most of the key components of the PC anticoagulant system had been identified [16]. Lately, the coagulation system has been refined because of new insights into the roles of the various cell types involved [17]. Hence, the initiation phase of coagulation is now known to commence on tissue factor (TF)-bearing cells such as macrophages, followed by an amplification phase involving platelet activation and activation of FV and FVIII. In the third phase, the propagation phase, bulk amounts of thrombin are generated on the surface of newly activated platelets [17]. The activity of the serine proteases generated during this sequence of reactions is under rigorous control, mediated by the TF pathway inhibitor and antithrombin; the latter a so-called serpin [18]. Likewise, APC and its co-factors, protein S and FV, regulate the activity of the activated forms of two homologous co-factors, FVIIIa and FVa [19]. In this review, we describe the post-translational modifications that occur in blood coagulation proteins and, where known, their functional implications. In the integrated defense system there are no borders between blood coagulation, the complement system, and the immune system. We have, however, taken a conservative approach and will deal only with those modifications that are found in the traditional coagulation factors, including the PC anticoagulant system and antithrombin. Seven years ago an excellent review of the same field was published [20].

A Central Role for GRB10 in Regulation of Islet Function in Man

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

Protein expression changes in ovarian cancer during the transition from benign to malignant.

Epithelial ovarian carcinoma has in general a poor prognosis since the vast majority of tumors are genomically unstable and clinically highly aggressive. This results in rapid progression of malignancy potential while still asymptomatic and thus in late diagnosis. It is therefore of critical importance to develop methods to diagnose epithelial ovarian carcinoma at its earliest developmental stage, that is, to differentiate between benign tissue and its early malignant transformed counterparts. Here we present a shotgun quantitative proteomic screen of benign and malignant epithelial ovarian tumors using iTRAQ technology with LC-MALDI-TOF/TOF and LC-ESI-QTOF MS/MS. Pathway analysis of the shotgun data pointed to the PI3K/Akt signaling pathway as a significant discriminatory pathway. Selected candidate proteins from the shotgun screen were further confirmed in 51 individual tissue samples of normal, benign, borderline or malignant origin using LC-MRM analysis. The MRM profile demonstrated significant differences between the four groups separating the normal tissue samples from all tumor groups as well as perfectly separating the benign and malignant tumors with a ROC-area of 1. This work demonstrates the utility of using a shotgun approach to filter out a signature of a few proteins only that discriminates between the different sample groups.

Generic workflow for quality assessment of quantitative label-free LC-MS analysis

As high‐resolution instruments are becoming standard in proteomics laboratories, label‐free quantification using precursor measurements is becoming a viable option, and is consequently rapidly gaining popularity. Several software solutions have been presented for label‐free analysis, but to our knowledge no conclusive studies regarding the sensitivity and reliability of each step of the analysis procedure has been described. Here, we use real complex samples to assess the reliability of label‐free quantification using four different software solutions. A generic approach to quality test quantitative label‐free LC‐MS is introduced. Measures for evaluation are defined for feature detection, alignment and quantification. All steps of the analysis could be considered adequately performed by the utilized software solutions, although differences and possibilities for improvement could be identified. The described method provides an effective testing procedure, which can help the user to quickly pinpoint where in the workflow changes are needed.

Automated Selected Reaction Monitoring Software for Accurate Label-Free Protein Quantification.

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5–19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

Streptococcus pyogenes in human plasma: adaptive mechanisms analyzed by mass spectrometry based proteomics.

Background: The human pathogen Streptococcus pyogenes adapts to vascular leakage at the site of infection. Results: S. pyogenes modifies the production of 213 in plasma determined using quantitative proteomics. Conclusion: The results clarify the function of HSA-binding proteins in S. pyogenes. Significance: Our data demonstrates the power of the quantitative mass spectrometry strategy to investigate bacterial adaptation to a given environment. Streptococcus pyogenes is a major bacterial pathogen and a potent inducer of inflammation causing plasma leakage at the site of infection. A combination of label-free quantitative mass spectrometry-based proteomics strategies were used to measure how the intracellular proteome homeostasis of S. pyogenes is influenced by the presence of human plasma, identifying and quantifying 842 proteins. In plasma the bacterium modifies its production of 213 proteins, and the most pronounced change was the complete down-regulation of proteins required for fatty acid biosynthesis. Fatty acids are transported by albumin (HSA) in plasma. S. pyogenes expresses HSA-binding surface proteins, and HSA carrying fatty acids reduced the amount of fatty acid biosynthesis proteins to the same extent as plasma. The results clarify the function of HSA-binding proteins in S. pyogenes and underline the power of the quantitative mass spectrometry strategy used here to investigate bacterial adaptation to a given environment.

Grading breast cancer tissues using molecular portraits

Tumor progression and prognosis in breast cancer patients are difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessments of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, determined using a novel affinity proteomics approach. We profiled 52 breast cancer tissue samples by combining nine antibodies and label-free LC-MS/MS, which generated detailed quantified proteomic maps representing 1,388 proteins. The results showed that we could define in-depth molecular portraits of histologically graded breast cancer tumors. Consequently, a 49-plex candidate tissue protein signature was defined that discriminated between histological grades 1, 2, and 3 of breast cancer tumors with high accuracy. Highly biologically relevant proteins were identified, and the differentially expressed proteins indicated further support for the current hypothesis regarding remodeling of the tumor microenvironment during tumor progression. The protein signature was corroborated using meta-analysis of transcriptional profiling data from an independent patient cohort. In addition, the potential for using the markers to estimate the likelihood of long-term metastasis-free survival was also indicated. Taken together, these molecular portraits could pave the way for improved classification and prognostication of breast cancer.

An Adaptive Alignment Algorithm for Quality-controlled Label-free LC-MS

Label-free quantification using precursor-based intensities is a versatile workflow for large-scale proteomics studies. The method however requires extensive computational analysis and is therefore in need of robust quality control during the data mining stage. We present a new label-free data analysis workflow integrated into a multiuser software platform. A novel adaptive alignment algorithm has been developed to minimize the possible systematic bias introduced into the analysis. Parameters are estimated on the fly from the data at hand, producing a user-friendly analysis suite. Quality metrics are output in every step of the analysis as well as actively incorporated into the parameter estimation. We furthermore show the improvement of this system by comprehensive comparison to classical label-free analysis methodology as well as current state-of-the-art software.

WNT5A-mediated β-catenin-independent signalling is a novel regulator of cancer cell metabolism.

WNT5A has been identified as an important ligand in the malignant progression of a number of tumours. Although WNT5A signalling is often altered in cancer, the ligand’s role as either a tumour suppressor or oncogene varies between tumour types and is a contemporary issue for investigators of β-catenin-independent WNT signalling in oncology. Here, we report that one of the initial effects of active WNT5A signalling in malignant melanoma cells is an alteration in cellular energy metabolism and specifically an increase in aerobic glycolysis. This was found to be at least in part due to an increase in active Akt signalling and lactate dehydrogenase (LDH) activity. The clinical relevance of these findings was strengthened by a strong correlation (P < 0.001) between the expression of WNT5A and LDH isoform V in a cohort of melanocytic neoplasms. We also found effects of WNT5A on energy metabolism in breast cancer cells, but rather than promoting aerobic glycolysis as it does in melanoma, WNT5A signalling increased oxidative phosphorylation rates in breast cancer cells. These findings support a new role for WNT5A in the metabolic reprogramming of cancer cells that is a context- dependent event.