Label-free Liquid Chromatography-mass Spectrometry Research Articles

Proteins involved in focal adhesion signaling pathways are differentially regulated in experimental branch retinal vein occlusion

Branch retinal vein occlusion (BRVO) is a common retinal vascular disease, but global protein changes following the condition remain largely unelucidated. To bring new insights into pathological processes and identify potential therapeutic targets, large-scale retinal protein changes following BRVO were studied by combining a porcine model of experimental BRVO with proteomic analysis by label-free liquid chromatography mass spectrometry. Among a total set of 1974 proteins, 52 significantly upregulated proteins and 10 significantly downregulated proteins were identified in retinas with BRVO after 15 days. Significantly upregulated proteins were involved in signaling pathways of focal adhesion via integrin and blood coagulation. Proteins involved in focal adhesion signaling included collagen α-2 chain, laminin subunit β-2, laminin subunit γ-1, lipocalin-7, nidogen-2, osteopontin, integrin-β, α-actinin-1, isoform 2 of α-actinin-1, talin-2 and filamin C. The identified proteins indicate that BRVO was associated with extracellular matrix remodeling processes. The present study identified focal adhesion signaling and ECM remodeling as important biological mechanisms to evaluate in the search for signaling pathways that promote neovascularisation and macular edema following BRVO.

Adaptation of the Rat Diaphragm Proteome in Response to Endurance Exercise Training

Mechanical ventilation is a lifesaving intervention for patients in respiratory failure. However, this leads to diaphragmatic atrophy and contractile dysfunction, termed ventilator-induced diaphragm dysfunction (VIDD). We recently discovered that endurance exercise training (ET) results in a diaphragmatic phenotype that resists VIDD. However, the cellular mechanisms responsible for ET-induced protection remain unknown. Therefore, ET provides a unique experimental tool to investigate novel biological targets to prevent VIDD. PURPOSE: Using a proteomics analysis, we examined the global protein expression changes in the diaphragm muscle following ET. METHODS: Female Sprague Dawley rats (∼3 months of age) were assigned to control (CON; N=8) or 2 weeks of endurance exercise-training (60 min/day at ∼ 70% VO2) (ET; N=8). Soluble proteins were extracted from diaphragm muscle, digested with trypsin, and analyzed using label-free liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: LC-MS/MS analysis of soluble proteins isolated from the diaphragm of CON and ET rats identified 1397 protein groups and label-free profiling was performed on 813 protein groups that had 3 or more unique peptides. The normalized abundance of 70 proteins was statistically (P<0.05) different between CON and ET samples. Importantly, our analysis revealed that ET leads to significant changes in the protein abundance of several potential cytoprotective proteins, which fall into the following categories, based upon biological functions: aerobic/fatty acid metabolism, redox regulation, stress adaptation, and calcium handling. Examples of proteins in each of these categories include long-chain-fatty-acid-CoA ligase 1 (+14%), microsomal glutathione S-transferase 3 (+18%), heat shock protein 70 kDa protein 1A/1B (+22%), and calsequestrin (-15%), respectively. CONCLUSION: These results uncover novel proteins that are differentially regulated with ET and highlight new areas of investigation to identify potential cytoprotective proteins that may serve as biological targets to prevent VIDD. Supported by the NIH R01 AR064189 awarded to SKP and alumni fellowship awarded to KJS.

Review, evaluation, and discussion of the challenges of missing value imputation for mass spectrometry-based label-free global proteomics.

In this review, we apply selected imputation strategies to label-free liquid chromatography-mass spectrometry (LC-MS) proteomics datasets to evaluate the accuracy with respect to metrics of variance and classification. We evaluate several commonly used imputation approaches for individual merits and discuss the caveats of each approach with respect to the example LC-MS proteomics data. In general, local similarity-based approaches, such as the regularized expectation maximization and least-squares adaptive algorithms, yield the best overall performances with respect to metrics of accuracy and robustness. However, no single algorithm consistently outperforms the remaining approaches, and in some cases, performing classification without imputation sometimes yielded the most accurate classification. Thus, because of the complex mechanisms of missing data in proteomics, which also vary from peptide to protein, no individual method is a single solution for imputation. On the basis of the observations in this review, the goal for imputation in the field of computational proteomics should be to develop new approaches that work generically for this data type and new strategies to guide users in the selection of the best imputation for their dataset and analysis objectives.

Maternal Immune Activation Induces Changes in Myelin and Metabolic Proteins, Some of Which Can Be Prevented with Risperidone in Adolescence

Background: Maternal infection is a risk factor for schizophrenia but the molecular and cellular mechanisms are not fully known. Myelin abnormalities are amongst the most robust neuropathological changes observed in schizophrenia, and preliminary evidence suggests that prenatal inflammation may play a role. Methods: Label-free liquid chromatography-mass spectrometry was performed on the prefrontal cortex (PFC) of adult rat offspring born to dams that were exposed on gestational day 15 to the viral mimic polyinosinic:polycytidylic acid [poly(I:C), 4 mg/kg] or saline and treated with the atypical antipsychotic drug risperidone (0.045 mg/kg) or saline in adolescence. Western blotting was employed to validate protein changes. Results: Over 1,000 proteins were quantified in the PFC with pathway analyses implicating changes in core metabolic pathways, following prenatal poly(I:C) exposure. Some of these protein changes were absent in the PFC of poly(I:C)-treated offspring that subsequently received risperidone treatment in adolescence. Particularly interesting reductions in the expression of the myelin-related proteins myelin basic protein isoform 3 (MBP1) and rhombex 29 were observed, which were reversed by risperidone treatment. Validation by Western blotting confirmed changes in MBP1 and mitogen-activated kinase 1 (MAPK1). Western blotting was extended to assess the MAPK signalling proteins due to their roles in inflammation, namely phosphorylated MAPK1 and phosphorylated MAPK-activated protein kinase 2. Both were upregulated by poly(I:C) treatment and reversed by risperidone treatment. Conclusions: Overall, our data suggest that maternal inflammation may contribute to an increased risk for schizophrenia through mechanisms involving metabolic function and myelin formation and that risperidone in adolescence may prevent or reverse such changes.

Dye-Free Porcine Model of Experimental Branch Retinal Vein Occlusion: A Suitable Approach for Retinal Proteomics.

Branch retinal vein occlusion induces complex biological processes in the retina that are generated by a multitude of interacting proteins. These proteins and their posttranslational modifications can effectively be studied using modern proteomic techniques. However, no method for studying large-scale protein changes following branch retinal vein occlusion has been available until now. Obtainment of retinal tissue exposed to branch retinal vein occlusion is only available through experimental animal models. Traditional models of experimental branch retinal vein occlusion require the use of Rose Bengal dye combined with argon laser photocoagulation. The use of Rose Bengal dye is problematic in proteomic studies as the dye can induce multiple protein modifications when irradiated. This paper presents a novel technique for proteomic analysis of porcine retinal tissue with branch retinal vein occlusion combining a dye-free experimental model with label-free liquid chromatography mass spectrometry based proteomics.

The positive is inside the negative: HER2-negative tumors can express the HER2 intracellular domain and present a HER2-positive phenotype

Overexpression of human epithelial growth factor receptor 2 (HER2) is a poor prognostic factor in breast cancer. HER2 is a transmembrane receptor comprising an extracellular domain (ECD), a single transmembrane domain, and an intracellular domain (ICD) with tyrosine-kinase activity. Receptor dimerization triggers pivotal effector pathways in cancer, such as phosphatidylinositol 3-kinase (PI3K) signaling. Currently, screening of HER2 in breast tumors for prognostic and therapeutic purposes involves immunohistochemical (IHC) phenotyping for the ECD, in which tumors with IHC scores below 2+ are reported as HER2-negative. We used a label-free liquid chromatography–mass spectrometry (LC–MS) proteomic approach to compare plasma samples from patients with HER2-positive breast tumors and patients with HER2-negative tumors. Patients with HER2-negative tumors expressed higher circulating levels of calpain-10 than patients with HER2-positive tumors. Calpains cleave HER2, releasing its ECD and transforming phenotypically positive tumors into phenotypically negative tumors. Therefore, we investigated the expression of the ICD in HER2-negative samples that overexpressed calpain-10. We found that 16% of HER2-negative tumors were positive for HER2-ICD, which was associated with circulating HER2-ECD. HER2 gene amplification was also observed in some HER2-negative tumors. Positive staining for the PI3K pathway was observed in the HER2-negative, ICD-positive tumors, similar to the HER2-positive cohort. Microarray analysis revealed that HER2-negative, ICD-positive samples clustered between HER2-positive tumors and triple-negative tumors. Survival analysis revealed that outcome in women with HER2-negative, ICD-positive tumors was better than in women bearing HER2-negative, ICD-negative (triple negative) tumors but was quite similar to HER2-positive tumors and worse than women with luminal A tumors. Moreover, in vitro analyses revealed that MDA-MB 231, a triple negative cell line, possesses calpain-10 and HER2-p-ICD up-regulation and blockage of calpain-10 activity promoted an increase in HER2-p-ICD and p-AKT levels, suggesting an increase in these pathways signaling. These data indicate that HER2-negative tumors with HER2-ICD positivity exhibit clinical behavior closer to that of HER2-positive tumors. This indicates a need for HER2-ICD screening when determining the molecular profile of breast tumors. These findings further indicate that lapatinib should be investigated as a target therapy for HER2-ICD-positive breast tumors.

Integrative proteomic analysis of the NMDA NR1 knockdown mouse model reveals effects on central and peripheral pathways associated with schizophrenia and autism spectrum disorders.

BackgroundOver the last decade, the transgenic N-methyl-D-aspartate receptor (NMDAR) NR1-knockdown mouse (NR1neo−/−) has been investigated as a glutamate hypofunction model for schizophrenia. Recent research has now revealed that the model also recapitulates cognitive and negative symptoms in the continuum of other psychiatric diseases, particularly autism spectrum disorders (ASD). As previous studies have mostly focussed on behavioural readouts, a molecular characterisation of this model will help to identify novel biomarkers or potential drug targets.MethodsHere, we have used multiplex immunoassay analyses to investigate peripheral analyte alterations in serum of NR1neo−/− mice, as well as a combination of shotgun label-free liquid chromatography mass spectrometry, bioinformatic pathway analyses, and a shotgun-based 40-plex selected reaction monitoring (SRM) assay to investigate altered molecular pathways in the frontal cortex and hippocampus. All findings were cross compared to identify translatable findings between the brain and periphery.ResultsMultiplex immunoassay profiling led to identification of 29 analytes that were significantly altered in sera of NR1neo−/− mice. The highest magnitude changes were found for neurotrophic factors (VEGFA, EGF, IGF-1), apolipoprotein A1, and fibrinogen. We also found decreased levels of several chemokines. Following this, LC-MSE profiling led to identification of 48 significantly changed proteins in the frontal cortex and 41 in the hippocampus. In particular, MARCS, the mitochondrial pyruvate kinase, and CamKII-alpha were affected. Based on the combination of protein set enrichment and bioinformatic pathway analysis, we designed orthogonal SRM-assays which validated the abnormalities of proteins involved in synaptic long-term potentiation, myelination, and the ERK-signalling pathway in both brain regions. In contrast, increased levels of proteins involved in neurotransmitter metabolism and release were found only in the frontal cortex and abnormalities of proteins involved in the purinergic system were found exclusively in the hippocampus.ConclusionsTaken together, this multi-platform profiling study has identified peripheral changes which are potentially linked to central alterations in synaptic plasticity and neuronal function associated with NMDAR-NR1 hypofunction. Therefore, the reported proteomic changes may be useful as translational biomarkers in human and rodent model drug discovery efforts.

Proteomic changes in serum of first onset, antidepressant drug-naïve major depression patients

Major depressive disorder (MDD) is a complex and multi-factorial disorder. Although genetic factors and other molecular aspects of MDD have been widely studied, the underlying pathological mechanisms are still mostly unknown. We sought to investigate the pathophysiology of MDD by identifying and characterising serum molecular differences and their correlation to symptom severity in first onset, antidepressant drug-naïve MDD patients. We performed an exploratory molecular profiling study on serum samples of MDD patients and controls using multiplex immunoassay and label-free liquid chromatography mass spectrometry in data independent mode (LC-MSE). We included two independent cohorts of first onset, antidepressant drug-naïve MDD patients (n=23 and 15) and matched controls (n=42 and 21) in our study in order to validate the results. The main outcome included the following list of circulatory molecules changing and/or correlating to symptom severity: angiotensin-converting enzyme, acute phase proteins (e.g. ferritin and serotransferrin), brain-derived neurotrophic factor, complement component C4-B, cortisol, cytokines (e.g. macrophage migration inhibitory factor and interleukin-16), extracellular newly identified receptor for advanced glycosylation end products-binding protein, growth hormone and superoxide dismutase-1. This study provides evidence of an increased pro-inflammatory and oxidative stress response, followed by a hyperactivation of the HPA-axis in the acute stages of first onset MDD, as well as a dysregulation in growth factor pathways. These findings help to elucidate MDD related pathways in more detail and further studies may lead to identification of novel drug targets, including components of the inflammatory and oxidative stress response.

Long-term Alterations in Maternal Plasma Proteome After sFlt1-induced Preeclampsia in Mice

Objective Preeclampsia is associated with long-term adverse maternal health, such as cardiovascular and metabolic diseases. The objective of this study was to determine whether preeclampsia in a well-characterized animal model that was induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt1) results in alterations in the maternal circulating proteome that persist long after delivery. Study Design CD-1 mice at day 8 of gestation were injected with adenovirus that carried sFlt1 or the murine immunoglobulin G2α Fc fragment as control. Depleted maternal plasma was analyzed 6 months after delivery by label-free liquid chromatography–mass spectrometry assay. The tandem mass spectrometry data were searched against a mouse database, and the resultant intensity data were used to compare abundance of proteins across disease/control plasma pool. Results were analyzed with ingenuity pathways analysis. Right-tailed Fisher exact test was used to calculate a probability value. Results Of 150 proteins that are common for both groups, ingenuity pathways analysis determined 105 proteins that were ready for analysis. Diseases and disorders analysis showed significant enrichment of proteins that are associated with cardiovascular disease. Within this cluster, the most abundant proteins were associated with vascular disease, atherosclerosis, and atherosclerotic lesions. Other top disease clusters were inflammatory response, organismal injury and abnormalities, and hematologic and metabolic disease. Conclusion Exposure to sFlt1-induced preeclampsia alters multiple biologic functions in mothers that persist later in life. Our results suggest that some of the long-term adverse outcomes that are associated with preeclampsia actually may be a consequence rather than a mere unmasking of an underlying predisposition. If similar results are found in humans, the development of preventive strategies for preeclampsia should also improve long-term maternal health.

Interactions among mitochondrial proteins altered in glioblastoma.

Mitochondrial dysfunction is putatively central to glioblastoma (GBM) pathophysiology but there has been no systematic analysis in GBM of the proteins which are integral to mitochondrial function. Alterations in proteins in mitochondrial enriched fractions from patients with GBM were defined with label-free liquid chromatography mass spectrometry. 256 mitochondrially-associated proteins were identified in mitochondrial enriched fractions and 117 of these mitochondrial proteins were markedly (fold-change ≥2) and significantly altered in GBM (p ≤ 0.05). Proteins associated with oxidative damage (including catalase, superoxide dismutase 2, peroxiredoxin 1 and peroxiredoxin 4) were increased in GBM. Protein–protein interaction analysis highlighted a reduction in multiple proteins coupled to energy metabolism (in particular respiratory chain proteins, including 23 complex-I proteins). Qualitative ultrastructural analysis in GBM with electron microscopy showed a notably higher prevalence of mitochondria with cristolysis in GBM. This study highlights the complex mitochondrial proteomic adjustments which occur in GBM pathophysiology.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-014-1430-5) contains supplementary material, which is available to authorized users.

Serum proteomic analysis identifies sex-specific differences in lipid metabolism and inflammation profiles in adults diagnosed with Asperger syndrome

BackgroundThe higher prevalence of Asperger Syndrome (AS) and other autism spectrum conditions in males has been known for many years. However, recent multiplex immunoassay profiling studies have shown that males and females with AS have distinct proteomic changes in serum.MethodsHere, we analysed sera from adults diagnosed with AS (males = 14, females = 16) and controls (males = 13, females = 16) not on medication at the time of sample collection, using a combination of multiplex immunoassay and shotgun label-free liquid chromatography mass spectrometry (LC-MSE). The main objective was to identify sex-specific serum protein changes associated with AS.ResultsMultiplex immunoassay profiling led to identification of 16 proteins that were significantly altered in AS individuals in a sex-specific manner. Three of these proteins were altered in females (ADIPO, IgA, APOA1), seven were changed in males (BMP6, CTGF, ICAM1, IL-12p70, IL-16, TF, TNF-alpha) and six were changed in both sexes but in opposite directions (CHGA, EPO, IL-3, TENA, PAP, SHBG). Shotgun LC-MSE profiling led to identification of 13 serum proteins which had significant sex-specific changes in the AS group and, of these, 12 were altered in females (APOC2, APOE, ARMC3, CLC4K, FETUB, GLCE, MRRP1, PTPA, RN149, TLE1, TRIPB, ZC3HE) and one protein was altered in males (RGPD4). The free androgen index in females with AS showed an increased ratio of 1.63 compared to controls.ConclusionTaken together, the serum multiplex immunoassay and shotgun LC-MSE profiling results indicate that adult females with AS had alterations in proteins involved mostly in lipid transport and metabolism pathways, while adult males with AS showed changes predominantly in inflammation signalling. These results provide further evidence that the search for biomarkers or novel drug targets in AS may require stratification into male and female subgroups, and could lead to the development of novel targeted treatment approaches.

Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatase 1: A Meaningful and Independent Marker to Predict Stroke in the Chinese Population

Label-free liquid chromatography-mass spectrometry (LC-MS) quantification methods have been described to determine serum proteins biomarkers in many diseases. Thus, the purpose of this study was to investigate the serum proteins biomarkers in the Chinese patients with acute ischemic stroke (AIS). In the study period, sera from 40 AIS patients and 40 normal cases were selected for screening study. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by label-free LC-MS/MS. The selected protein associations with disease risk were further evaluated by enzyme-linked immunosorbent assay (ELISA) testing of the remaining stroke cases and controls. Its value for biomarkers diagnosis was appreciated through receiver operating curve (ROC). Patients versus control levels differences were suggested for 19 proteins (nominal P < 0.05) for stroke, with three proteins having a false discovery rate <0.05. The association of Phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 (SHIP-1) with stroke (P < 0.001) was confirmed using ELISA in replication studies. Based on the ROC curve, the optimal cut-off value of serum SHIP-1 levels for diagnosis of stroke was projected to be 1,550 pg/ml, which yielded a sensitivity of 77.5% and a specificity of 88.3%. In multivariate analysis, there was an increased risk of AIS associated with SHIP-1 levels ≥ 1,550 pg/ml (OR 4.28, 95% CI: 1.97-8.96) after adjusting for possible confounders. The discovery and replication studies presented here show SHIP-1 to be a risk marker for AIS in the Chinese population, which appears to be a novel finding.

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody-antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immunoglobulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody-antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metalloproteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15-25% of patient samples by enzyme-linked immunosorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future.

SAT0246 Proteomic analysis of synovial tissue: A unique tool to predict response to anti-TNF-α therapy in patients with inflammatory arthritis

BackgroundInflammatory arthritis, which includes rheumatoid arthritis (RA) and psoriatic arthritis (PsA), is a leading cause of joint deformity, disability and reduced quality of life with a high economic cost [1]....

Long-term alterations in maternal plasma proteome after sFlt1–induced preeclampsia in mice

Preeclampsia is associated with long-term adverse maternal health, such as cardiovascular and metabolic diseases. The objective of this study was to determine whether preeclampsia in a well-characterized animal model that was induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt1) results in alterations in the maternal circulating proteome that persist long after delivery. CD-1 mice at day 8 of gestation were injected with adenovirus that carried sFlt1 or the murine immunoglobulin G2α Fc fragment as control. Depleted maternal plasma was analyzed 6 months after delivery by label-free liquid chromatography-mass spectrometry assay. The tandem mass spectrometry data were searched against a mouse database, and the resultant intensity data were used to compare abundance of proteins across disease/control plasma pool. Results were analyzed with ingenuity pathways analysis. Right-tailed Fisher exact test was used to calculate a probability value. Of 150 proteins that are common for both groups, ingenuity pathways analysis determined 105 proteins that were ready for analysis. Diseases and disorders analysis showed significant enrichment of proteins that are associated with cardiovascular disease. Within this cluster, the most abundant proteins were associated with vascular disease, atherosclerosis, and atherosclerotic lesions. Other top disease clusters were inflammatory response, organismal injury and abnormalities, and hematologic and metabolic disease. Exposure to sFlt1-induced preeclampsia alters multiple biologic functions in mothers that persist later in life. Our results suggest that some of the long-term adverse outcomes that are associated with preeclampsia actually may be a consequence rather than a mere unmasking of an underlying predisposition. If similar results are found in humans, the development of preventive strategies for preeclampsia should also improve long-term maternal health.

An Integrated Proteomics Reveals Pathological Mechanism of Honeybee (Apis cerena) Sacbrood Disease

Viral diseases of honeybees are a major challenge for the global beekeeping industry. Chinese indigenous honeybee (Apis cerana cerana, Acc) is one of the major Asian honeybee species and has a dominant population with more than 3 million colonies. However, Acc is frequently threatened by a viral disease caused by Chinese sacbrood virus (CSBV), which leads to fatal infections and eventually loss of the entire colony. Nevertheless, knowledge on the pathological mechanism of this deadly disease is still unknown. Here, an integrated gel-based and label-free liquid chromatography-mass spectrometry (LC-MS) based proteomic strategy was employed to unravel the molecular event that triggers this disease, by analysis of proteomics and phosphoproteomics alterations between healthy and CSBV infected worker larvae. There were 180 proteins and 19 phosphoproteins which altered their expressions after the viral infection, of which 142 proteins and 12 phosphoproteins were down-regulated in the sick larvae, while only 38 proteins and 7 phosphoproteins were up-regulated. The infected worker larvae were significantly affected by the pathways of carbohydrate and energy metabolism, development, protein metabolism, cytoskeleton, and protein folding, which were important for supporting organ generation and tissue development. Because of abnormal metabolism of these pathways, the sick larvae fail to pupate and eventually death occurs. Our data, for the first time, comprehensively decipher the molecular underpinnings of the viral infection of the Acc and are potentially helpful for sacbrood disease diagnosis and medicinal development for the prevention of this deadly viral disease.

Adaptive Changes in the Neuronal Proteome: Mitochondrial Energy Production, Endoplasmic Reticulum Stress, and Ribosomal Dysfunction in the Cellular Response to Metabolic Stress

Impaired energy metabolism in neurons is integral to a range of neurodegenerative diseases, from Alzheimer's disease to stroke. To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, we have defined the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to a metabolic challenge of oxygen glucose deprivation (OGD) in vitro. A total of 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry. The levels of 130 proteins were significantly increased (P<0.01) after OGD and the levels of 63 proteins were significantly decreased (P<0.01) while expression of the majority of proteins (765) was not altered. Network analysis identified novel protein–protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins was associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Our investigation of the global cellular response to a metabolic challenge clearly shows the considerable adaptive capacity of the proteome to a slowly evolving metabolic challenge.

Protein phosphorylation patterns in serum from schizophrenia patients and healthy controls

Most proteomic studies to date have attempted to identify changes in protein levels without considering the effects of post-translational modifications (PTM). However, characteristic changes of PTM such as phosphorylation could be biologically informative, as these can give insights into disease and drug mechanisms of action at the functional level. With this in mind, we have conducted a comparative proteomic and phosphoproteomic analysis of blood sera from 20 antipsychotic-naïve schizophrenia patients and 20 matched healthy controls. We used immobilised metal ion affinity chromatography (IMAC) for enrichment of phosphoproteins combined with label-free liquid chromatography-mass spectrometry (LC-MS(E)) for identification and measurement of protein and phosphoprotein levels. The LC-MS(E) analysis of both IMAC-fractions resulted in identification of 35 proteins with altered levels in schizophrenia. Analysis of the enriched fraction resulted in identification of 72 phosphoproteins with altered phosphorylation patterns. Of these, 59 showed changes in phosphorylation only, with no overall change in protein levels. This study provided evidence that schizophrenia patients feature serum abnormalities in phosphorylation of proteins involved in acute phase response and coagulation pathways. Further studies of such phosphorylation-specific changes could lead to a better understanding of the molecular aetiology of schizophrenia, and provide a means of biomarker identification for clinical studies. This article is part of a Special Issue entitled: Integrated omics.

Identification of proteomic signatures associated with depression and psychotic depression in post-mortem brains from major depression patients

Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis of post-mortem dorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

Using a spike-in experiment to evaluate analysis of LC-MS data

BackgroundRecent advances in liquid chromatography-mass spectrometry (LC-MS) technology have led to more effective approaches for measuring changes in peptide/protein abundances in biological samples. Label-free LC-MS methods have been used for extraction of quantitative information and for detection of differentially abundant peptides/proteins. However, difference detection by analysis of data derived from label-free LC-MS methods requires various preprocessing steps including filtering, baseline correction, peak detection, alignment, and normalization. Although several specialized tools have been developed to analyze LC-MS data, determining the most appropriate computational pipeline remains challenging partly due to lack of established gold standards.ResultsThe work in this paper is an initial study to develop a simple model with "presence" or "absence" condition using spike-in experiments and to be able to identify these "true differences" using available software tools. In addition to the preprocessing pipelines, choosing appropriate statistical tests and determining critical values are important. We observe that individual statistical tests could lead to different results due to different assumptions and employed metrics. It is therefore preferable to incorporate several statistical tests for either exploration or confirmation purpose.ConclusionsThe LC-MS data from our spike-in experiment can be used for developing and optimizing LC-MS data preprocessing algorithms and to evaluate workflows implemented in existing software tools. Our current work is a stepping stone towards optimizing LC-MS data acquisition and testing the accuracy and validity of computational tools for difference detection in future studies that will be focused on spiking peptides of diverse physicochemical properties in different concentrations to better represent biomarker discovery of differentially abundant peptides/proteins.