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Objective: Extracellular vesicles (EVs) have the potential to act as intercellular communicators. The aims were to characterize circulating EVs in patients with pulmonary arterial hypertension (PAH) and to explore whether these EVs contribute to endothelial activation and angiogenesis. Approach and Results: Patients with PAH (n=70) and healthy controls (HC; n=20) were included in this cross-sectional study. EVs were characterized and human pulmonary endothelial cells (hPAECs) were incubated with purified EVs. Endothelial cell activity and proangiogenic markers were analyzed. Tube formation analysis was performed for hPAECs, and the involvement of PSGL-1 (P-selectin glycoprotein ligand 1) was evaluated. The numbers of CD62P + , CD144 + , and CD235a EVs were higher in blood from PAH compared with HC. Thirteen proteins were differently expressed in PAH and HC EVs, where complement fragment C1q was the most significantly elevated protein ( P =0.0009) in PAH EVs. Upon EVs-internalization in hPAECs, more PAH compared with HC EVs evaded lysosomes ( P P P Conclusions: Patients with PAH have higher levels of EVs harboring increased amounts of angiogenic proteins, which induce activation of hPAECs and in vitro angiogenesis. These effects were partly because of platelet-derived EVs evasion of lysosomes upon internalization within hPAEC and through possible involvement of P-selectin-PSGL-1 pathway.

Acute pulmonary embolism (PE) is a common emergency with a high morbidity and mortality. Most clinical presentations are non-specific and there is a lack of suitable biomarkers for PE. For example, the traditional D-dimer tests shows a rather high sensitivity for PE, but yet a rather low positive predictive value due to its lack of specificity. Research on novel biomarkers for PE is thus of interest to improve early diagnostics and reduce the number of unnecessary computed tomography pulmonary angiogram (CTPA) scans performed. In this study we evaluate the feasibility to use label-free quantitative proteomics to discover potential biomarkers for acute PE and to monitor changes in proteins levels in PE patients over time. Blood was collected from 8 patients with CTPA verified PE and from 8 patients presenting with same symptoms but with a negative CTPA. The samples were analyzed by liquid chromatography-mass spectrometry and thirteen protein concentrations were found to be significantly changed in PE patients compared to the CTPA negative controls. This exploratory study shows that proteomic analysis can be used to identify potential biomarkers for PE as well as to monitor changes of protein levels over time. The complement proteins play a part in PE but further studies are needed to clarify their specific role in the pathophysiological process and to look for more specific proteins.

Alzheimer’s disease (AD) is characterized by impaired protein homeostasis leading to amyloid-beta (Aβ) amyloidosis. Here, we show that autophagy is similarly inhibited in AD brains and App knock-in AD mouse models. To determine how pathologies translate to cerebrospinal fluid (CSF), label-free mass spectrometry of mouse CSF was used. This identified autophagy-related extracellular matrix (ECM) protein decorin as significantly and similarly increased in App knock-in mice and cognitively normal human subjects with abnormal CSF-amyloid. Notably, a switch from negative to positive correlation of CSF-decorin and CSF-amyloid occurs in cognitively normal subjects when CSF-amyloid becomes abnormal, indicating an early change in CSF-decorin induced by Aβ amyloidosis. In App knock-in mice increased CSF-decorin correlated with accentuated decorin expression in choroid plexus and decreased interneuronal decorin. Furthermore, decorin activates neuronal autophagy-lysosomal system by enhancing lysosomal degradation. Therefore, decorin is a potential CSF biomarker that reflects ECM and autophagy alterations in early AD Aβ amyloidosis.

BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long‐term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3.ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM.

Objective Identifying molecular changes that contribute to the onset and progression of Huntington's disease (HD) is of importance for the development and evaluation of potential therapies. Methods We conducted an unbiased mass‐spectrometry proteomic analysis on the cerebrospinal fluid of 12 manifest HD patients (ManHD), 13 pre‐manifest (preHD), and 38 controls. A biologically plausible and significant possible biomarker was validated in samples from a separate cohort of patients and controls consisting of 23 ManHD patients and 23 controls. Results In ManHD compared to preHD, 10 proteins were downregulated and 43 upregulated. Decreased levels of proenkephalin (PENK) and transthyretin were closely linked to HD symptom severity, whereas levels of 15 upregulated proteins were associated with symptom severity. The decreased PENK levels were replicated in the separate cohort where absolute quantitation was performed. Conclusions We hypothesize that declining PENK levels reflect the degeneration of medium spiny neurons (MSNs) that produce PENK and that assays for PENK may serve as a surrogate marker for the state of MSNs in HD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1—non-muscle invasive bladder cancer (NMIBC), and T2/T3—muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers., Open access funding provided by Uppsala University., This work was supported by Magnus Bergvall Foundation (S.B.L, 2017-02330, 2018-02726, 2019-03296), Clas Groschinsky memory foundation (S.B.L., M1603, M1742), and Swedish Foundation for Strategic research (S.B.L., SB16-0039). A.V. would like to acknowledge the Ministry of Science and Innovation for a “Juan de la Cierva” postdoctoral Grant.

The muscle RING-finger protein-1 (MuRF1) is an E3 ubiquitin ligase expressed in skeletal and cardiac muscle tissues and it plays important roles in muscle remodeling. Upregulation of MuRF1 gene transcription participates in skeletal muscle atrophy, on contrary downregulation of protein expression leads to cardiac hypertrophy. MuRF1 gene point mutations have been found to generate protein aggregate myopathies defined as muscle disorder characterized by protein accumulation in muscle fibers. We have discovered that MuRF1 turned out to be also a target for a new post-translational modification arbitrated by conjugation of SUMO1 and it is mediated by the SUMO ligases E2 UBC9 and the E3 PIASγ/4. SUMOylation takes place at lysine 238 localized at the second coiled-coil protein domain that is required for efficient substrate interaction for polyubiquitination. We provided evidence that SUMOylation is essential for MuRF1 nuclear translocation and its mitochondria accumulation is enhanced in hyperglycemic conditions delivering a stabilization of the overall SUMOylated proteins in cultured myocytes. Thus, our findings add this SUMO1 post-translational modification as a new concept to understand muscle disorders related to the defect in MuRF1 activity.

The main cause of trigeminal neuralgia (TN) is compression of a blood vessel at the root entry zone of the trigeminal nerve. However, a neurovascular conflict does not seem to be the only etiology and other mechanisms are implicated in the development of the disease. We hypothesized that TN patients may have distinct protein expression in the CSF. In this study, lumbar CSF from TN patients (n = 17), scheduled to undergo microvascular decompression, and from controls (n = 20) was analyzed and compared with in depth mass spectrometry TMTbased quantitative proteomics. We identified 2552 unique proteins, of which 46 were significantly altered (26 increased, and 20 decreased, q-value.05) in TN patients compared with controls. An over-representation analysis showed proteins involved in high-density lipoprotein, such as Apolipoprotein A4, Apolipoprotein M, and Apolipoprotein A1, and the extracellular region, including proteins involved in the complement cascade to be over-represented. We conclude that TN patients have distinct protein expression in the CSF compared to controls. The pathophysiological background of the protein alterations found in this study warrants further investigation in future studies. PERSPECTIVE: In this article, cerebrospinal fluid from patients with trigeminal neuralgia was analyzed using in depth shotgun proteomics, revealing 46 differentially expressed proteins compared to controls. Among these, apolipoproteins and proteins involved in the complement system were elevated and significantly over-represented, implying an inflammatory component in the pathophysiology of the disease.

In the field of Alzheimer’s disease, there is an urgent need for novel analytical tools to identify disease-specific biomarkers and to evaluate therapeutics. Preclinical trials commonly employ amyloid beta (Aβ) peptide signatures as a read-out. In this paper, we report a simplified and detailed protocol for robust immunoprecipitation of Aβ in brain tissue prior to mass spectrometric detection exemplified by a study using transgenic mice. The established method employed murine monoclonal and rabbit polyclonal antibodies and was capable of yielding well-reproducible peaks of high intensity with low background signal intensities corresponding to various Aβ forms.

Molecular screening programs for cervical cancer detect the presence of human papilloma virus (HPV) in cell material or vaginal fluids. Persistent infection with high-risk HPV is a necessary pre-requisite, but the majority of infections do not lead to pathological states. Additional biomarkers are needed to increase the specificity of the molecular tests. Here, we have investigated the possibility of detecting protein biomarkers using mass spectrometry from dried self-sampled cervico–vaginal fluid deposited on FTA cards. We found significant intra-individual correlations (p &lt, 2.2 × 10−16), although heterogenous protein profiles were obtained between individuals. Out of 3699 proteins found in total, 169 were detected in at least 95% of the samples. Using a discovery/replication design, 18 proteins were found to be significant in the discovery cohort, with higher values in those cases compared to controls. All of these were found to also have higher levels among the cases in the replication cohort, with one protein (DEAD-Box Helicase) remaining statistically significant. Finally, a predictive 7-protein multivariate model was developed with a sensitivity and specificity of 0.90 and 0.55, respectively. Our results demonstrate that robust measurements of protein biomarkers can be obtained from self-sampled dried CVF and that these could be used to predict cervical cancer pre-stages.

Background Alzheimer's disease (AD) is diagnosed based on a clinical evaluation as well as analyses of classical biomarkers: Aβ42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF). Although the sensitivities and specificities of the classical biomarkers are fairly good for detection of AD, there is still a need to develop novel biochemical markers for early detection of AD. Objective We explored if integration of novel proteins with classical biomarkers in CSF can better discriminate AD from non-AD subjects. Methods We applied ELISA, mass spectrometry, and multivariate modeling to investigate classical biomarkers and the CSF proteome in subjects (n = 206) with 76 AD patients, 74 mild cognitive impairment (MCI) patients, 11 frontotemporal dementia (FTD) patients, and 45 non-dementia controls. The MCI patients were followed for 4-9 years and 21 of these converted to AD, whereas 53 remained stable. Results By combining classical CSF biomarkers with twelve novel markers, the area of the ROC curves (AUROCS) of distinguishing AD and MCI/AD converters from non-AD were 93% and 96%, respectively. The FTDs and non-dementia controls were identified versus all other groups with AUROCS of 96% and 87%, respectively. Conclusions Integration of new and classical CSF biomarkers in a model-based approach can improve the identification of AD, FTD, and non-dementia control subjects.

Extracellular vesicles (EVs), including exosomes and larger microvesicles, have been implicated to play a role in several conditions, including Alzheimer’s disease (AD). Since the EV content mirrors the intracellular environment, it could contribute with important information about ongoing pathological processes and may be a useful source for biomarkers, reflecting the disease progression. The aim of the present study was to analyze the protein content of EVs specifically released from a mixed co-culture of primary astrocytes, neurons, and oligodendrocytes treated with synthetic amyloid-β (Aβ42) protofibrils. The EV isolation was performed by ultracentrifugation and validated by transmission electron microscopy. Mass spectrometry analysis of the EV content revealed a total of 807 unique proteins, of which five displayed altered levels in Aβ42 protofibril exposed cultures. The most prominent protein was apolipoprotein E (apoE), and by western blot analysis we could confirm a threefold increase of apoE in EVs from Aβ42 protofibril exposed cells, compared to unexposed cells. Moreover, immunoprecipitation studies demonstrated that apoE was primarily situated inside the EVs, whereas immunocytochemistry indicated that the EVs most likely derived from the astrocytes and the neurons in the culture. The identified Aβ-induced sorting of apoE into EVs from cultured neuroglial cells suggests a possible role for intercellular transfer of apoE in AD pathology and encourage future studies to fully elucidate the clinical relevance of this event.

We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

The article examines the establishment of media market in Georgia – one of republics of the former Soviet Union. Today Georgia is one of the leading countries of the Caucasian region recognized in various international ratings. Georgia holds the first place in the index of freedom of mass media in the region, which demonstrates a real democratic transformation in the country. The article considers national legislation regulating mass media and analyses the key media market players. While describing the major stages of the establishment of the Georgian radio and television, authors pay attention to the fact that some media genres, for example, radio-performances, were very popular in all post-Soviet space and abroad. Nowadays Georgia is a country with the developed media market with leading world players present in all media segments (television, radio, press, cable television, satellite television, internet, blogosphere, independent TV studios). In addition, citizens of Georgia are active Internet, social media and blog users. The authors conclude that despite certain existing difficulties and problems of the Georgian media market, the country has good prospects of sectoral development in the case of a political will.

Mammalian brain is challenging to study due to its heterogeneity and complexity. However, recent advances in molecular imaging, genomics and proteomics have contributed significantly to achieve insights into molecular basis of brain function and pathogenesis of neurological disorders. Efficient sample preparation is an integral part of a successful mass spectrometry (MS)-based proteomics. Apart from the identification, quantification of proteins is needed to investigate the alterations between proteome profiles from different sample sets. Therefore, this thesis investigates optimizing and application of the MS compatible sample preparation techniques for the identification and quantification of proteins from brain tissue.The central objective of this thesis was (i) to improve the extraction of proteins as well as membrane proteins (MPs) from the brain tissue and (ii) to apply the optimized method along with the stable isotope dimethyl labeling (DML) and label-free (LF) MS approaches for the relative quantification of the brain proteome profiles during neurological conditions such as Alzheimer’s disease (AD) and traumatic brain injury (TBI). First study described in this thesis is focused on the qualitative aspects for the brain tissue sample preparation. The optimized extraction buffers from first study containing n-octyl-β-glucopyranside or triton X-114 were used in the further quantitative studies to extract the proteins from patient (AD or TBI) and control human brain samples. Triton X-114 has additional advantage of separating MPs into a micellar phase. Therefore we also investigated the possibility to apply this in combination with DML quantitation approach for enrichment of low abundant MPs from AD brains.AD and TBI causes severe socio-economic burden on the society and therefore there is a need to develop diagnostic markers to detect the early changes in the pathology of the disease. Analytical tools and techniques applied and discussed in this thesis for neuroproteomics applications proved to be powerful and reliable for analyzing complex biological samples to generate high-throughput screening and unbiased identification and quantitation of disease-specific proteins that are of great importance in understanding the disease pathology.

Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.

Neurodegenerative disorders are often defined pathologically by the presence of protein aggregates, such as amyloid plaques composed of β-amyloid (Aβ) peptide in Alzheimer's disease. Such aggregates are the result of abnormal protein accumulation and may lead to neuronal dysfunction and cell death. In this study, APPSWE transgenic mice (Tg2576), which overexpress the Swedish mutated form of human amyloid precursor protein (APP), were used to study the brain proteome associated with amyloid plaque deposition. The major aim of the study was to map and compare the Tg2576 model brain proteome profiles during pathology progression using a shotgun approach based on label free quantification with mass spectrometry. Overall, 1085 proteins were identified and longitudinally quantified. Principal component analysis (PCA) showed the appearance of the pathology onset between twelve and fifteen months, correlating with sharp amyloid plaque accumulation within the same ages. Cluster analysis followed by protein-protein interaction analysis revealed an age-dependent decrease in mitochondrial protein expression. We identified 57 significantly affected mitochondrial proteins, several of which have been reported to alter expression in neurological diseases. We also found ten proteins that are upregulated early in the amyloid driven pathology progression with high confidence, some of which are directly involved in the onset of mitochondrial apoptosis and may represent potential markers for use in human neurological diseases prognosis. Our results further contribute to identifying common pathological pathways involved in both aging and progressive neurodegenerative disorders enhancing the understanding of disease pathogenesis.

In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first "shotgun"-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20-30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16-23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17-31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.

In this study, temperature-induced phase fractionation also known as cloud-point extraction (CPE) with the nonionic surfactant Triton X-114 was used to simultaneously extract hydrophobic and hydrophilic proteins from porcine brain tissue. Various protein precipitation/delipidation procedures were investigated to efficiently remove lipids and detergents while retaining maximum protein recoveries. The best performing delipidation method was then used in combination with CPE to compare three different mass spectrometry (MS) based "bottom-up" proteomic approaches for protein analysis of the porcine brain. In the first approach, the intact proteins were initially separated by one-dimensional (1D) gel electrophoresis. The excised protein bands were digested with trypsin, and the peptides were separated by reversed phase nanoliquid chromatography (RP-nanoLC) followed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis. The other bottom-up proteomic approaches were based on first enzymatical digestion of the proteins followed by RP-nanoLC separation in combination with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) or on the combination of in-solution isoelectric focusing (IEF) with ESI-nanoLC-MS/MS of the IEF separated peptides. In total, we found and unambiguously identified 331 unique proteins. The overlap between different techniques was about 10%, showing that the use of multiple proteomic approaches is beneficial to yield a better coverage of the proteome. Furthermore, the overlap between the CPE extracted hydrophilic and hydrophobic proteins was rather small (9-16%), indicating an efficient sample preparation technique to extract and separate hydrophilic and hydrophobic proteins from brain tissue. The percentage of identified membrane proteins was 27%, which is in accordance to the fact that about one-third of all genes in various organisms encode for this class of proteins. The results indicate that cloud point extraction is a promising sample preparation tool, which allows simultaneous in depth studies of brain derived membrane proteins as well as hydrophilic proteins. This technique can be very useful when studying human central nervous system (CNS) tissue or animal models of neurological diseases.

Alzheimer's disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer's disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer's disease patients and non-demented controls to identify potential biomarkers for Alzheimer's disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer's disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p

Neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases.11 This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Integral membrane proteins (MPs), such as transporters, receptors, and ion channels, are of great interest because of their participation in various vital cellular functions including cell-cell interactions, ion transport, and signal transduction. However, studies of MPs are complicated because of their hydrophobic nature, heterogeneity, and low abundance. Cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was performed to simultaneously extract and phase separate hydrophobic and hydrophilic proteins from Alzheimer's disease (AD) and unaffected control brain tissue. Quantitative proteomics analysis of temporal neocortex samples of AD patients and controls was performed using a shotgun approach based on stable isotope dimethyl labeling (DML) quantification technique followed by nanoLC-MS/MS analysis. A total of 1096 unique proteins were identified and quantified, with 40.3 % (211/524) predicted as integral MPs with at least one transmembrane domain (TMD) found in the detergent phase, and 10 % (80/798) in the detergent-depleted phase. Among these, 62 proteins were shown to be significantly altered (p-value

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive muscle paralysis. Currently clinical tools for ALS diagnostics do not perform well enough and their improvement is needed. The objective of this study was to identify specific protein alterations related to the development of ALS using tiny muscle biopsies. We applied a shotgun proteomics and quantitative dimethyl labeling in order to analyze the global changes in human skeletal muscle proteome of ALS versus healthy subjects for the first time. 235 proteins were quantified and 11 proteins were found significantly regulated in ALS muscles. These proteins are involved in muscle development and contraction, metabolic processes, enzyme activity, regulation of apoptosis and transport activity. In order to eliminate a risk to confuse ALS with other denervations, muscle biopsies of patients with postpolio syndrome and Charcot–Marie–Tooth disease (negative controls) were compared to those of ALS and controls. Only few proteins significantly regulated in ALS patients compared to controls were affected differently in negative controls. These proteins (BTB and kelch domain-containing protein 10, myosin light chain 3, glycogen debranching enzyme, transitional endoplasmic reticulum ATPase), individually or as a panel, could be selected for estimation of ALS diagnosis and development. Biological significance ALS is a devastating neurodegenerative disease, and luckily, very rare: only one to two people out of 100,000 develop ALS yearly. This fact, however, makes studies of ALS very challenging since it is very difficult to collect the representative set of clinical samples and this may take up to several years. In this study we collected the muscle biopsies from 12 ALS patients and compared the ALS muscle proteome against the one from control subjects. We suggested the efficient method for such comprehensive quantitative analysis by LC–MS and performed it for the first time using human ALS material. This gel- and antibody-free method can be widely applied for muscle proteome studies and has been used by us for revealing of the specific protein alterations associated with ALS.

The complicated secondary molecular and cellular mechanisms following traumatic brain injury (TBI) are still not fully understood. In the present study, we have used mass spectrometry to identify injury specific proteins in an in vitro model of TBI. A standardized injury was induced by scalpel cuts through a mixed cell culture of astrocytes, oligodendrocytes and neurons. Twenty-four hours after the injury, cell culture medium and whole-cell fractions were collected for analysis. We found 53 medium proteins and 46 cell fraction proteins that were specifically expressed after injury and the known function of these proteins was elucidated by an extensive literature survey. By using time-lapse microscopy and immunostainings we could link a large proportion of the proteins to specific cellular processes that occur in response to trauma; including cell death, proliferation, lamellipodia formation, axonal regeneration, actin remodeling, migration and inflammation. A high percentage of the proteins uniquely expressed in the medium after injury were actin-related proteins, which normally are situated intracellularly. We show that two of these, ezrin and moesin, are expressed by astrocytes both in the cell culture model and in mouse brain subjected to experimental TBI. Interestingly, we found many inflammation-related proteins, despite the fact that cells were present in the culture. This study contributes with important knowledge about the cellular responses after trauma and identifies several potential cell-specific biomarkers.

Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ-FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-β-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.