Person:

Steen, Hanno

The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or ‘phospho-form’—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the ‘phospho-form distribution’: the relative amount of each of the 2(^n) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods—western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.

Background: Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry. Results: DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles. Conclusion: We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the (APC/C^{Cdh1}) complex, but not by the (APC/C^{Cdc20}) complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this FLEXIQinase assay we combined our Full-Length Expressed Stable Isotope-labeled Protein for Quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multi-kinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n×80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal Kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3β activity and permitting an advanced model for GSK3β-mediated signaling.

Objectives: The secretin-stimulated endoscopic pancreatic function test (ePFT) allows for the safe collection of gastroduodenal and pancreatic fluid from the duodenum. We test the hypothesis that these endoscopically collected fluids have different proteomes. As such, we aim to show that the ePFT method can be used to collect fluid enriched in pancreatic proteins to test for pancreatic function. Methods: Gastroduodenal and pancreatic fluid were collected sequentially from chronic pancreatitis patients undergoing an ePFT. Proteins from each fluid type were extracted using previously published optimized methods and subjected to GeLC-MS/MS analysis for protein identification and bioinformatics analysis. Results: Mass spectrometry analysis identified proteins that were exclusive in either gastroduodenal (46) or pancreatic fluid (234). Subsequent quantitative analysis revealed proteins that were differentially abundant with statistical significance. As expected, proteolytic enzymes and protease inhibitors were among the differentially detected proteins. The proteases pepsinogens and gastrin were enriched in gastroduodenal fluid, while common pancreatic enzymes (e.g., aminopeptidase N, chymotrypsin C, elastase-3A, trypsin, and carboxypeptidase A1, and elastase 2B) were found in greater abundance in pancreatic fluid. Similarly for protease inhibitors, members of the cystatin family were exclusive to gastroduodenal fluid, while serpins A11, B4, and D1 were exclusive to pancreatic fluid. Conclusions: We have shown that ePFT collection coupled with mass spectrometry can be used to identify differentially detected proteins in gastroduodenal and pancreatic fluids. The data obtained using GeLC-MS/MS techniques provide further evidence supporting the feasibility of using ePFT-collected fluid to study specific diseases of the upper gastrointestinal tract, such as chronic pancreatitis.

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease.

Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which ∼30 - 40% were common to all treatment conditions and ∼5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix™). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles, paralleling responses to adjuvant-containing vaccines in vivo. Age-specific in vitro modeling coupled with proteomics may provide fresh insight into the ontogeny of adjuvant action thereby informing targeted adjuvanted vaccine development for distinct age groups.

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. Absence of definitive diagnostic markers limits the accuracy of clinical evaluations of suspected KD with significant increases in morbidity. In turn, incomplete understanding of its molecular pathogenesis hinders the identification of rational targets needed to improve therapy. We used high-accuracy mass spectrometry proteomics to analyse over 2000 unique proteins in clinical urine specimens of patients with KD. We discovered that urine proteomes of patients with KD, but not those with mimicking conditions, were enriched for markers of cellular injury such as filamin and talin, immune regulators such as complement regulator CSMD3, immune pattern recognition receptor muclin, and immune cytokine protease meprin A. Significant elevations of filamin C and meprin A were detected in both the serum and urine in two independent cohorts of patients with KD, comprised of a total of 236 patients. Meprin A and filamin C exhibited superior diagnostic performance as compared to currently used markers of disease in a blinded case-control study of 107 patients with suspected KD, with receiver operating characteristic areas under the curve of 0.98 (95% confidence intervals [CI] of 0.97–1 and 0.95–1, respectively). Notably, meprin A was enriched in the coronary artery lesions of a mouse model of KD. In all, urine proteome profiles revealed novel candidate molecular markers of KD, including filamin C and meprin A that exhibit excellent diagnostic performance. These disease markers may improve the diagnostic accuracy of clinical evaluations of children with suspected KD, lead to the identification of novel therapeutic targets, and allow the development of a biological classification of Kawasaki disease.

Only a small fraction of the mammalian genome codes for messenger RNAs destined to be translated into proteins, and it is generally assumed that a large portion of transcribed sequences - including introns and several classes of non-coding RNAs (ncRNAs) do not give rise to peptide products. A systematic examination of translation and physiological regulation of ncRNAs has not been conducted. Here, we use computational methods to identify the products of non-canonical translation in mouse neurons by analyzing unannotated transcripts in combination with proteomic data. This study supports the existence of non-canonical translation products from both intragenic and extragenic genomic regions, including peptides derived from anti-sense transcripts and introns. Moreover, the studied novel translation products exhibit temporal regulation similar to that of proteins known to be involved in neuronal activity processes. These observations highlight a potentially large and complex set of biologically regulated translational events from transcripts formerly thought to lack coding potential.

Objectives: Chronic pancreatitis (CP) may be difficult to diagnose in early stages. We aimed to measure pancreatic juice (PJ) prostaglandin E2 (PGE2) concentrations to determine whether they are elevated in CP and improve diagnosis of early disease. Methods: We measured PJ PGE2 in 10 patients with established CP, 25 patients who met criteria for “minimal change” chronic pancreatitis (MCCP), and 10 normal control participants. Results: Median PJ PGE2 was elevated in CP (307 pg/ml, IQR (249–362)) and MCCP (568 pg/ml, (418–854)) compared with normal controls (104 pg/ml, (68–206)) (P≤ 0.001). Area under receiving operator curve (AUROC) for diagnosis of CP and MCCP was 0.9 and 0.62, respectively, for PJ bicarbonate concentration alone; AUROC was 1.0 and 0.94 for the combination of PJ bicarbonate and PGE2 concentrations. Conclusions: PJ PGE2 appears to be a biomarker for CP and is elevated in both established and “minimal change” chronic pancreatitis.