Glycolysis in hepatic stellate cells coordinates fibrogenic extracellular vesicle release spatially to amplify liver fibrosis.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 (RAB31) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

PP2A catalytic subunit alpha is critically required for CD8+ T cell homeostasis and anti-bacterial responses.

While the functions of tyrosine phosphatases in T cell biology have been extensively studied, our knowledge on the contribution of serine/threonine phosphatases in T cells remains poor. Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine phosphatases. It is important in thymocyte development and CD4+ T cell differentiation. Utilizing a genetic model in which its catalytic subunit alpha isoform (PP2A Cα) is deleted in T cells, we investigated its contribution to CD8+ T cell homeostasis and effector functions. Our results demonstrate that T cell intrinsic PP2A Cα is critically required for CD8+ T cell homeostasis in secondary lymphoid organs and intestinal mucosal site. Importantly, PP2A Cα deficient CD8+ T cells exhibit reduced proliferation and survival. CD8+ T cell anti-bacterial response is strictly dependent on PP2A Cα. Expression of Bcl2 transgene rescues CD8+ T cell homeostasis in spleens, but not in intestinal mucosal site, nor does it restore the defective anti-bacterial responses. Finally, proteomics and phosphoproteomics analyses reveal potential targets dependent on PP2A Cα, including mTORC1 and AKT. Thus, PP2A Cα is a key modulator of CD8+ T cell homeostasis and effector functions.

Proteomic and phosphoproteomic analyses of myectomy tissue reveals difference between sarcomeric and genotype-negative hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetically heterogenous condition with about half of cases remaining genetically elusive or non-genetic in origin. HCM patients with a positive genetic test (HCMSarc) present earlier and with more severe disease than those with a negative genetic test (HCMNeg). We hypothesized these differences may be due to and/or reflect proteomic and phosphoproteomic differences between the two groups. TMT-labeled mass spectrometry was performed on 15 HCMSarc, 8 HCMNeg, and 7 control samples. There were 243 proteins differentially expressed and 257 proteins differentially phosphorylated between HCMSarc and HCMNeg. About 90% of pathways altered between genotypes were in disease-related pathways and HCMSarc showed enhanced proteomic and phosphoproteomic alterations in these pathways. Thus, we show HCMSarc has enhanced proteomic and phosphoproteomic dysregulation observed which may contribute to the more severe disease phenotype.

Optimizing single cell proteomics using trapped ion mobility spectrometry for label-free experiments.

Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Furthermore, trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated in data-dependent acquisition mode (DDA-PASEF) allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating the ion flux in TIMS affects the overall performance of proteome profiling. However, the effect of TIMS settings on the analysis of low-input samples has been less investigated. Thus, we sought to optimize the conditions of TIMS with regard to ion accumulation/ramp times and ion mobility range for low-input samples. We observed that an ion accumulation time of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 V s cm-2 resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We used these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1116, and 1651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from a low number of cells was sufficient to delineate several essential metabolic pathways and the T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that such an approach could be applied to label-free analysis of single cells obtained from clinically relevant samples.

An atlas of the bone marrow bone proteome in patients with dysproteinemias.

Multiple myeloma (MM) bone disease is a significant cause of morbidity but there is a paucity of data on the impact of malignant plasma cells on adjacent trabecular bone within the BM. Here, we characterize the proteome of trabecular bone tissue from BM biopsies of 56 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM), newly diagnosed (NDMM), relapsed MM (RMM), and normal controls. Proteins involved in extracellular matrix (ECM) formation and immunity pathways were decreased in SMM and active MM. Among the proteins most decreased were immunoglobulins, type IV collagen, and TIMP3, suggesting increased immunoparesis and decreased ECM remodelling within trabecular bone. Proteins most increased in SMM/MM were APP (enhances osteoclast activity), ENPP1 (enhances bone mineralization), and MZB1 (required for normal plasmablast differentiation). Pathway analyses showed that proteins involved in gamma -carboxylation, a pathway implicated in osteocalcin function, osteoblast differentiation, and normal hematopoiesis, were also overexpressed in SMM/MM. This study is the first comprehensive proteomic atlas of the BM bone proteome in dysproteinemias. We identify new key proteins and pathways for MM bone disease and potentially impaired hematopoiesis, and show for the first time that gamma -carboxylation pathways are increased in the bone tissue of SMM/MM.

Multi-Omic Architecture of Obstructive Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy. Currently, hypertrophy pathways responsible for HCM have not been fully elucidated. Their identification could serve as a nidus for the generation of novel therapeutics aimed at halting disease development or progression. Herein, we performed a comprehensive multi-omic characterization of hypertrophy pathways in HCM. METHODS: Flash-frozen cardiac tissues were collected from genotyped HCM patients (n=97) undergoing surgical myectomy and tissue from 23 controls. RNA sequencing and mass spectrometry-enabled deep proteome and phosphoproteomic assessment were performed. Rigorous differential expression, gene set enrichment, and pathway analyses were performed to characterize HCM-mediated alterations with emphasis on hypertrophy pathways. RESULTS: We identified transcriptional dysregulation with 1246 (8%) differentially expressed genes and elucidated downregulation of 10 hypertrophy pathways. Deep proteomic analysis identified 411 proteins (9%) that differed between HCM and controls with strong dysregulation of metabolic pathways. Seven hypertrophy pathways were upregulated with antagonistic upregulation of 5 of 10 hypertrophy pathways shown to be downregulated in the transcriptome. Most upregulated hypertrophy pathways encompassed the rat sarcoma-mitogen-activated protein kinase signaling cascade. Phosphoproteomic analysis demonstrated hyperphosphorylation of the rat sarcoma-mitogen-activated protein kinase system suggesting activation of this signaling cascade. There was a common transcriptomic and proteomic profile regardless of genotype. CONCLUSIONS: At time of surgical myectomy, the ventricular proteome, independent of genotype, reveals widespread upregulation and activation of hypertrophy pathways, mainly involving the rat sarcoma-mitogen-activated protein kinase signaling cascade. In addition, there is a counterregulatory transcriptional downregulation of the same pathways. Rat sarcoma-mitogen-activated protein kinase activation may serve a crucial role in hypertrophy observed in HCM.

Four-dimensional proteomics analysis of human cerebrospinal fluid with trapped ion mobility spectrometry using PASEF.

A quadrupole time-of-flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation-serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four-dimensional (4D) features comprising of elution time, collision cross section (CCS), mass-to-charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap-derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library-free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.

Molecular signatures of inherited and acquired sporadic late onset nemaline myopathies.

Acquired sporadic late onset nemaline myopathy (SLONM) and inherited nemaline myopathy (iNM) both feature accumulation of nemaline rods in muscle fibers. Unlike iNM, SLONM is amenable to therapy. The distinction between these disorders is therefore crucial when the diagnosis remains ambiguous after initial investigations. We sought to identify biomarkers facilitating this distinction and to investigate the pathophysiology of nemaline rod formation in these different disorders. Twenty-two muscle samples from patients affected by SLONM or iNM underwent quantitative histological analysis, laser capture microdissection for proteomic analysis of nemaline rod areas and rod-free areas, and transcriptomic analysis. In all iNM samples, nemaline rods were found in subsarcolemmal or central aggregates, whereas they were diffusely distributed within muscle fibers in most SLONM samples. In SLONM, muscle fibers harboring nemaline rods were smaller than those without rods. Necrotic fibers, increased endomysial connective tissue, and atrophic fibers filled with nemaline rods were more common in SLONM. Proteomic analysis detected differentially expressed proteins between nemaline rod areas and rod-free areas, as well as between SLONM and iNM. These differentially expressed proteins implicated immune, structural, metabolic, and cellular processes in disease pathophysiology. Notably, immunoglobulin overexpression with accumulation in nemaline rod areas was detected in SLONM. Transcriptomic analysis corroborated proteomic findings and further revealed substantial gene expression differences between SLONM and iNM. Overall, we identified unique pathological and molecular signatures associated with SLONM and iNM, suggesting distinct underlying pathophysiological mechanisms. These findings represent a step towards enhanced diagnostic tools and towards development of treatments for SLONM.

13C15N: glucagon-based novel isotope dilution mass spectrometry method for measurement of glucagon metabolism in humans.

BACKGROUND: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. METHODS: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. RESULTS: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. CONCLUSIONS: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

Proteome-Wide Response of Dormant Caryopses of the Weed, Avena fatua, After Colonization by a Seed-Decay Isolate of Fusarium avenaceum.

Promoting seed decay is an ecological approach to reducing weed persistence in the soil seedbank. Previous work demonstrated that Fusarium avenaceum F.a.1 decays dormant Avena fatua (wild oat) caryopses and induces several defense enzyme activities in vitro. The objectives of this study were to obtain a global perspective of proteins expressed after F.a.1-caryopsis colonization by conducting proteomic evaluations on (i) leachates, soluble extrinsic (seed-surface) proteins released upon washing caryopses in buffer and (ii) proteins extracted from whole caryopses; interactions with aluminum (Al) were also evaluated in the latter study because soil acidification and associated metal toxicity are growing problems. Of the 119 leachate proteins classified as defense/stress, 80 were induced or repressed. Defense/stress proteins were far more abundant in A. fatua (35%) than in F.a.1 (12%). Avena defense/stress proteins were also the most highly regulated category, with 30% induced and 35% repressed by F.a.1. Antifungal proteins represented 36% of Avena defense proteins and were the most highly regulated, with 36% induced and 37% repressed by F.a.1. These results implicate selective regulation of Avena defense proteins by F.a.1. Fusarium proteins were also highly abundant in the leachates, with 10% related to pathogenicity, 45% of which were associated with host cell wall degradation. In whole caryopsis extracts, fungal colonization generally resulted in induction of a similar set of Avena proteins in the presence and absence of Al. Results advance the hypothesis that seed decay pathogens elicit intricate and dynamic biochemical responses in dormant seeds.

Proteomic profiling of sporadic late-onset nemaline myopathy.

OBJECTIVE: To define the proteomic profile of sporadic late-onset nemaline myopathy (SLONM) and explore its pathogenesis. METHODS: We performed mass spectrometry on laser-dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL). RESULTS: There were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T-cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers. CONCLUSION: Our findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.

The proteome of distal nerves: implication in delayed repair and poor functional recovery.

Chronic denervation is one of the key factors that affect nerve regeneration. Chronic axotomy deteriorates the distal nerve stump, causes protein changes, and renders the microenvironment less permissive for regeneration. Some of these factors/proteins have been individually studied. To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect, we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model. Four rats that received immediate repair after sciatic nerve transection served as control, whereas four rats in the experimental group (chronic denervation) had their sciatic nerve repaired after a 12-week delay. All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins. Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels. NanoLC-MS/MS analysis was applied to the gels. Protein expression levels of nerves on the surgery side were compared to those on the contralateral side. Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed. All the differentially expressed proteins in both groups were further stratified according to the biological processes. A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration. Ingenuity Pathway Analysis (IPA) software was used for pathway analysis. The results showed that 709 differentially expressed proteins were identified in the delayed repair group, with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified. The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated. IPA revealed that protective pathways such as LXR/RXR, acute phase response, RAC, ERK/MAPK, CNTF, IL-6, and FGF signaling were inhibited in the delayed repair group, whereas three detrimental pathways, including the complement system, PTEN, and apoptosis signaling, were activated. An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types. The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins. The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.

Analysis of the polycystin complex (PCC) in human urinary exosome-like vesicles (ELVs).

The polycystin-1 (PC1), polycystin-2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome-like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C-terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C-terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C-terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is  > 2 MDa in size and that N-terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.

Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins.

Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impacts the sensitivity of the measurement. We therefore applied a high-resolution orbitrap mass spectrometer to the analysis of [ring-(13)C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high-resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. At higher enrichment levels, enrichment measurements from the orbitrap platform showed significant correlation (R (2) > 0.5) with those of the triple quadrupole platform. Together, these results indicate that high-resolution MS platforms such as the orbitrap are not only as capable of performing isotope enrichment measurements as the more commonly preferred triple quadrupole instruments, but offer unparalleled advantages in terms of mass accuracy and sensitivity in the presence of similar-mass contaminants.

Identification of Biomarkers for PKD1 Using Urinary Exosomes.

Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075).

Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine.

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.

Using the Clinical Prevention and Population Health Curriculum Framework to encourage curricular change.

The Healthy People Curriculum Task Force was established in 2002 to encourage implementation of Healthy People 2010 Objective 1.7: "To increase the proportion of schools of medicine, schools of nursing and health professional training schools whose basic curriculum for healthcare providers includes the core competencies in health promotion and disease prevention." In 2004, the Task Force published a Clinical Prevention and Population Health Curriculum Framework ("Framework") to help each profession assess and develop more robust approaches to this content in their training. During the 6 years since the publication of the Framework, the Task Force members introduced and disseminated it to constituents, facilitated its implementation at member schools, integrated it into initiatives that would influence training across schools, and adapted and applied the Framework to meet the data needs of the Healthy People 2010 Objective 1.7. The Framework has been incorporated into initiatives that help promote curricular change, such as accreditation standards and national board examination content, and efforts to disseminate the experiences of peers, expert recommendations, and activities to monitor and update curricular content. The publication of the revised Framework and the release of Healthy People 2020 (and the associated Education for Health Framework) provide an opportunity to review the efforts of the health professions groups to advance the kind of curricular change recommended in Healthy People 2010 and Healthy People 2020 and to appreciate the many strategies required to influence health professions curricula.

Bi-Linear Regression for O Quantification: Modeling across the Elution Profile.

MOTIVATION: Interpreting and quantifying labeled mass-spectrometry data is complex and requires automated algorithms, particularly for large scale proteomic profiling. Here, we propose the use of bi-linear regression to quantify relative abundance across the elution profile in a unified model. The bi-linear regression model takes advantage of the fact that while peptides differ in overall abundance across the elution profile multiplicatively, the relative abundance between the mixed samples remains constant across the elution profile. We describe how to apply bi-linear regression models to (18)O stable-isotope labeled data, which allows for the direct comparison of two samples simultaneously. Interpretation of model parameters is also discussed. The incorporation rate of the labeling isotope is estimated as part of the modeling process and can be used as a measure of data quality. Application is demonstrated in a controlled experiment as well as in a complex mixture. RESULTS: Bi-linear regression models allow for more precise and accurate estimates of abundance, in comparison to methods that treat each spectrum independently, by taking into account the abundance of the molecule throughout the entire elution profile, with precision increased by one-to-two orders of magnitude.

Discovery of naturally processed and HLA-presented class I peptides from vaccinia virus infection using mass spectrometry for vaccine development.

An important approach for developing a safer smallpox vaccine is to identify naturally processed immunogenic vaccinia-derived peptides rather than live whole vaccinia virus. We used two-dimensional liquid chromatography coupled to mass spectrometry to identify 116 vaccinia peptides, encoded by 61 open reading frames, from a B-cell line (homozygous for HLA class I A*0201, B*1501, and C*03) after infection with vaccinia virus (Dryvax). Importantly, 68 of these peptides are conserved in variola, providing insight into the peptides that induce protection against smallpox. Twenty-one of these 68 conserved peptides were 11 amino acids long or longer, outside of the range of most predictive algorithms. Thus, direct identification of naturally processed and presented HLA peptides gives important information not provided by current computational methods for identifying potential vaccinia epitopes.

A method for automatically interpreting mass spectra of 18O-labeled isotopic clusters.

16O/18O labeling is one differential proteomics technology among many that promises diagnostic and prognostic biomarkers of disease. Although the incorporation of 18O in the C-terminal carboxyl group during endoproteinase digestion in the presence of H2 18O makes the process of labeling facile, the ease and effectiveness of label incorporation have in some regards been outweighed by the difficulties in interpreting the resulting spectra. Complex isotope patterns result from the composition of unlabeled (18O(0)), singly labeled (18O(1)), and doubly labeled species (18O(2)) as well as contributions from the naturally occurring isotopes (e.g. 13C and 15N). Moreover because labeling is enzymatic, the number of 18O atoms incorporated can vary from peptide to peptide. Finally it is difficult to distinguish highly up-regulated from highly down-regulated or C-terminal peptides. We have developed an algorithm entitled regression analysis applied to mass spectrometry (RAAMS) that automatically, rapidly, and confidently interprets spectra of 18O-labeled peptides without requiring chemical composition information derived from product ion spectra. The algorithm is able to measure the effective 18O incorporation rate due to variable enzyme substrate specificity of the pseudosubstrate during the isotope exchange reaction and corrects for the 18O(0) abundance that remains in the labeled sample when using a two-step digestion/labeling procedure. We have also incorporated a method for distinguishing pure 18O(0) from pure 18O(2) peptides utilizing impure H2 18O. The algorithm operates on centroided peak lists and is therefore very fast: nine chromatograms of, on average, 1,168 spectra and containing, on average, 6,761 isotopic clusters were interpreted in, on average, 45 s per chromatogram. RAAMS is fast enough (average, 38 ms/spectrum) to allow the possibility of performing information-dependent MS/MS on a chromatographic time scale on species exceeding predetermined ratio thresholds. We describe in detail the operation of the algorithm and demonstrate its use on datasets with known and unknown ratios.