Nanoflow Liquid Chromatography-tandem Mass Research Articles

In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells.

Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC\u2013MS/MS

Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC–MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC–MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC–MS/MS is suitable for a broad range of proteomic applications.

Preliminary Investigation on Difference of Protein Compositions Between DC2.4 Cells and Their Derived Exosomes by nanoLC-MS/MS

To preliminarily investigate the differences of protein composition between immature dendritic cells (DC2.4) and their derived exosomes (DC-Exo) using a relatively rapid and sample-saving method based on nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The supernatant of DC2.4 cells culture medium was collected and gradient centrifugation was applied to primarily extract and isolate DC-Exo; then sucrose density gradient ultracentrifugation was adopted to purify the DC-Exo. Bradford protein assay was used to determine the total protein content of the purified DC-Exo, and dynamic light scattering and transmission electron microscope were conducted to characterize the morphology and size distribution of the DC-Exo. Afterwards, protein samples including DC2.4 cells and DC-Exo were prepared by FASP enzymolysis method. Samples were performed nanoLC-MS/MS assay. The μLPickUp sample loading mode was used and only 1 μg of protein sample was required for each assay. The phase of Transport liquid and Micro A were both 0.05% trifluoroacetic acid-2% acetonitrile (ACN) aq. ( V/ V). Acclaim ® PepMap RSLC column was used to separate sample compositions and the gradient elute was adopted where the mobile phase consisted of (A) 0.1% formic acid (FA) and (B) 0.08% FA-80% ACN aq. ( V/ V) with flow rate of 0.3 μL/min. Positive APCI nanospray interface was used and "one-drive-ten" schema was set to collect primary information. The collected data was then searched and matched based on Uniport Mouse Fasta file as protein database in this case, and the re-annotated data was further sorted out and analyzed. In the current study, relatively high yield of DC-Exo samples with sizes of 40-200 nm were obtained. The lyophilized protein samples prepared by FASP method could be loaded directly after redissolution, and only 1 μg of protein sample is required. The annotated results showed that DC2.4 cells contained 998 kinds of proteins, among which 227 were highly expressed and 535 were unique; while DC-Exo contained only 348 types of proteins, among which 18 were uniquely and highly expressed. There were 306 kinds of consensus proteins in both DC2.4 cells and DC-Exo, among them 7 kinds were highly expressed. The nanoLC-MS/MS method developed in this study only requires very small amount of protein samples, and it could primarily differentiate the protein compositions between DC2.4 cells and their derived exosomes rapidly.

Mapping Relative Differences in Human Salivary Gland Secretions by Dried Saliva Spot Sampling and nanoLC-MS/MS.

Dried saliva spot sampling is a minimally invasive technique for the spatial mapping of salivary protein distribution in the oral cavity. In conjunction with untargeted nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis, DSS is used to compare the proteomes secreted by unstimulated parotid and submandibular/sublingual salivary glands. Two hundred and twenty proteins show a statistically significant association with parotid gland secretion, while 30 proteins are at least tenfold more abundant in the submandibular/sublingual glands. Protein identifications and label-free quantifications are highly reproducible across the paired glands on three consecutive days, enabling to establish the core proteome of glandular secretions categorized into eight salivary protein groups according to their biological functions. The data suggest that the relative contributions of the salivary glands fine-tune the biological activity of human saliva via medium-abundant proteins. A number of biomarker candidates for Sjögren's syndrome are observed among the gland-specifically expressed proteins, which indicates that glandular origin is an important factor to consider in salivary biomarker discovery.

Proteomic analysis of liver tissue from dogs with chronic hepatitis.

Chronic hepatitis is the most common hepatic disease in dogs. Copper accumulation is an important cause of chronic hepatitis in dogs; however, the etiology in most dogs cannot be determined. Clinical signs of chronic hepatitis are often non-specific; therefore, this disease is frequently diagnosed in an advanced stage that makes successful intervention less likely. Early diagnosis of chronic hepatitis in dogs would thus be beneficial. The identification of proteins that are differentially expressed in dogs with chronic hepatitis could contribute to the development of novel diagnostic markers for this disease and provide insight into its pathogenesis. The objective of this study was to identify novel proteins that are differentially expressed in the liver of dogs with chronic hepatitis. Hepatic tissue was collected from 8 healthy dogs during ovariohysterectomy and from 8 dogs with histologically confirmed chronic hepatitis. The proteome of the liver samples was extracted by mechanical disruption and detergent-based cell lysis and differentially labeled prior to analysis by 2-dimensional fluorescence difference gel electrophoresis. Spots with an absolute fold change value > 2.0 were selected for further analysis. Protein identification was achieved by nanoflow liquid chromatography tandem mass spectrometry. Differential expression of select proteins was validated by Western blot. Five protein spots were differentially expressed between patients with chronic hepatitis and healthy control dogs. From these 5 protein spots 11 proteins were identified. Differential expression of cytokeratin 18 and annexin 5 were confirmed by Western blot analysis. Differential protein expression was shown between dogs with chronic hepatitis and healthy control dogs. Upregulation of cytokeratin 18 in chronic hepatitis may suggest increased hepatocellular apoptosis and necrosis, whereas upregulation of annexin 5A suggests increased hepatocellular apoptosis. Further studies are needed to determine whether either protein has diagnostic utility.

Proteomic profile of root canal contents in teeth with post‐treatment endodontic disease

To characterize the proteome of 20 root canals in teeth with post-treatment endodontic disease using mass spectrometry and to correlate the identified proteins with clinical features. Twenty patients with radiographic evidence of apical periodontitis and need for root canal re-treatment were selected. Samples from the root canal contents were collected and processed using two-dimensional capillary nano-flow liquid chromatography and electrospray ionization tandem mass spectrometry. The acquired spectra were separately searched against specific protein database. The results obtained were analysed using descriptive statistics. Additionally, Pearson's chi-square test or one-sided Fisher's exact test, as appropriate, was chosen to examine the null hypothesis that there is no relationship between each clinical feature and the presence of specific microbial or human proteins. Significance levels were set at 5% (P<0.05). A total of 1153 human and 720 microbial UniProt accession numbers corresponding to proteins were recovered. The greater prevalence of proteins was related to biological functions, such as cellular and metabolic processes. A considerable number of microbial proteins with clinical relevance functions, such as pathogenesis/virulence, proteolysis, cell adhesion and drug resistance, were detected. Common endodontic pathogens related to post-treatment endodontic disease such as Enterococcus spp., Propionibacterium spp. and Streptococcus spp. were associated with 23, 40 and 94 distinct proteins, respectively. As for human proteins, many factors related to the immune system process were detected. No significant correlations were found between microbial and human proteins and the clinical features investigated (P>0.05). A considerable number of microbial and human proteins were identified using proteomic analyses, being mainly related to processes indicating cell viability. No significant correlation was found between proteins and clinical features. These findings suggest a network of important microbial pathogenic functions that may be responsible for the host immune system response.

Stability of glyoxal- and methylglyoxal-modified hemoglobin on dried blood spot cards as analyzed by nanoflow liquid chromatography tandem mass spectrometry

Blood sampling by the dried blood spot (DBS) technique has become commonly applied in newborn screening. It is often used for analysis of small molecules, such as metabolites. Recently, DBS sampling has been applied for quantification of post-translational protein modifications. Glyoxal and methylglyoxal are two simple oxoaldehydes released from glycated proteins in the Maillard reaction. They are widely distributed in the environment (e.g. cigarette smoke) and found in foods and beverages. Glyoxal and methylglyoxal are shown to react with biomolecules including DNA and proteins. In this laboratory, we previously identified the sites of modification by these two oxoaldehydes in human hemoglobin and found that the extents of modification at certain sites of lysine and arginine residues are significantly higher in type 2 diabetes mellitus patients than in nondiabetic individuals. In this study, we examine the stability of these modifications of hemoglobin stored on DBS cards at room temperature or 4 °C in the ambient air. After hemoglobin was extracted from the DBS cards, it was digested by trypsin and analyzed by nanoflow liquid chromatography coupled with nanospray ionization tandem mass spectrometry. The results show that the extents of all these PTMs are stable within 14 and 21 days when stored on DBS at room temperature and at 4 °C, respectively. Extraction of globin from DBS cards is mostly advantageous for hemolytic blood samples. This assay is sensitive as only a quarter of a DBS card containing ca. 12 μL of blood is required. Thus, it is practically useful to measure the extents of glyoxal- and methylglyoxal-induced hemoglobin modifications from DBS cards.

Age-Associated Methylation in Human Hemoglobin and Its Stability on Dried Blood Spots As Analyzed by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

Methylation of biomolecules is involved in many important biological processes. The contributing methylating agents arise from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as a molecular dosimeter for monitoring chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin by nanoflow liquid chromatography tandem mass spectrometry under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in hemoglobin were quantified in nonsmoking subjects at various ages in this study. Among the nine methylation sites, we found that the extents of methylation were significantly higher in elderly subjects at the N-terminal and His-20 of α-globin, and at the N-terminal and Glu-26 of β-globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. We also examined the stability of methylated and ethylated hemoglobin when stored on dried blood spot cards. The extents of these modifications on hemoglobin are stable for at least 4 weeks stored at room temperature. Our results suggest that age should be considered as a factor when measuring hemoglobin methylation and that dried blood spot is a valuable biomonitoring technique for hemoglobin modifications in epidemiological studies.

Metabolomic Analysis of Glioma Cells Using Nanoflow Liquid Chromatography-Tandem Mass Spectrometry.

Mass spectrometry (MS)-based techniques have been finding utility as sensitive, high throughput metabolite analysis tools for complex biological samples. We describe here a nanoflow liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system we developed and applied to metabolic profiling of human cells. Metabolites are extracted from cells using methanol, and filtered through a C18 StageTip to remove large particles. Metabolite samples are separated by HPLC at a flow rate of 400-500 nl/min, then analyzed in both positive and negative ion modes in an LTQ-Orbitrap MS. Metabolite identification and differential analysis are performed using commercial or open source software. Protocols outlined in this chapter describe how nano-LC-MS can be applied to investigate metabolic profiling with limited biomass amount.

A Stable Isotope Dilution Nanoflow Liquid Chromatography Tandem Mass Spectrometry Assay for the Simultaneous Detection and Quantification of Glyoxal-Induced DNA Cross-Linked Adducts in Leukocytes from Diabetic Patients

Glyoxal (gx) is a bifunctional electrophile capable of cross-linking DNA. Although it is present in foods and from the environment, endogenous formation of glyoxal occurs through metabolism of carbohydrates and oxidation of lipids and nucleic acids. Plasma concentrations of glyoxal are elevated in in diabetes mellitus patients compared to nondiabetics. The most abundant 2'-deoxyribonucleoside adducts cross-linked by glyoxal are dG-gx-dC, dG-gx-dA, and dG-gx-dG. These DNA cross-links can be mutagenic by damaging the integrity of the DNA structure. Herein, we developed a highly sensitive and specific assay for the simultaneous detection and quantification of the dG-gx-dC and dG-gx-dA cross-links based on stable isotope dilution (SID) nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selected reaction monitoring mode and using a triple quadrupole mass spectrometer. The entire assay procedure involved addition of the stable isotope standards [15N5]dG-gx-dC and [15N5]dG-gx-dA as internal standards, enzyme hydrolysis to release the cross-links as nucleosides, enrichment by a reversed-phase solid-phase extraction column, and nanoLC-NSI/MS/MS analysis. The detection limit is 0.19 amol for dG-gx-dC and 0.89 amol for dG-gx-dA, which is 400 and 80 times more sensitive, respectively, than capillary LC-NSI/MS/MS assay of these adducts. The lower limit of quantification was 94 and 90 amol for dG-gx-dC and dG-gx-dA, respectively, which is equivalent to 0.056 and 0.065 adducts in 108 normal nucleotides in 50 μg of DNA. In type 2 diabetes mellitus (T2DM) patients (n = 38), the levels of dG-gx-dC and dG-gx-dA in leukocyte DNA were 1.94 ± 1.20 and 2.10 ± 1.77 in 108 normal nucleotides, respectively, which were significantly higher than those in nondiabetics (n = 39: 0.83 ± 0.92 and 1.05 ± 0.99 in 108 normal nucleotides, respectively). Excluding the factor of smoking, an exogenous source of glyoxal, levels of these two cross-linked adducts were found to be significantly higher in nonsmoking T2DM patients than in nonsmoking control subjects. Furthermore, the levels of dG-gx-dC and dG-gx-dA correlated with HbA1c with statistical significance. To our best knowledge, this is the first report of the identification and quantification of glyoxal-derived cross-linked DNA adducts in human leukocyte DNA and their association with T2DM. This SID nanoLC-NSI/MS/MS assay is highly sensitive and specific and it requires only 50 μg of leukocyte DNA isolated from 2-3 mL of blood to accurately quantify these two cross-linked adducts simultaneously. Our assay thus provides a useful biomarker for the evaluation of glyoxal-derived DNA damage.

Analysis of Chlorination, Nitration, and Nitrosylation of Tyrosine and Oxidation of Methionine and Cysteine in Hemoglobin from Type 2 Diabetes Mellitus Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

The post-translational modification (PTM) of proteins by endogenous reactive chlorine, nitrogen, and oxygen species is implicated in certain pathological conditions, including diabetes mellitus. Evidence showed that the extents of modifications on a number of proteins are elevated in diabetic patients. Measuring modification on hemoglobin has been used to monitor the extent of exposure. This study develops an assay for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin and to examine whether the level of any of these modifications is higher in poorly controlled type 2 diabetic mellitus patients. This mass spectrometry-based assay used the bottom-up proteomic strategy. Due to the low amount of endogenous modification, we first characterized the sites of chlorination at tyrosine in hypochlorous acid-treated hemoglobin by an accurate mass spectrometer. The extents of chlorination, nitration, and oxidation of a total of 12 sites and types of modifications in hemoglobin were measured by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry under the selected reaction monitoring mode. Relative quantification of these PTMs in hemoglobin extracted from blood samples shows that the extents of chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in diabetic patients (n = 19) than in nondiabetic individuals (n = 18). After excluding the factor of smoking, chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in the nonsmoking diabetic patients (n = 12) than in normal nonsmoking subjects (n = 11). Multiple regression analysis performed on the combined effect of age, body-mass index (BMI), and HbA1c showed that the diabetes factor HbA1c contributes significantly to the extent of chlorination at α-Tyr-24 in nonsmokers. In addition, age contributes to oxidation at α-Met-32 significantly in all subjects and in nonsmokers. These results suggest the potential of using chlorination at α-Tyr-24-containing peptide to evaluate protein damage in nonsmoking type 2 diabetes mellitus.

Evaluation of treadmill exercise effect on muscular lipid profiles of diabetic fatty rats by nanoflow liquid chromatography-tandem mass spectrometry.

We compare comprehensive quantitative profiling of lipids at the molecular level from skeletal muscle tissues (gastrocnemius and soleus) of Zucker diabetic fatty rats and Zucker lean control rats during treadmill exercise by nanoflow liquid chromatography–tandem mass spectrometry. Because type II diabetes is caused by decreased insulin sensitivity due to excess lipids accumulated in skeletal muscle tissue, lipidomic analysis of muscle tissues under treadmill exercise can help unveil the mechanism of lipid-associated insulin resistance. In total, 314 lipid species, including phospholipids, sphingolipids, ceramides, diacylglycerols (DAGs), and triacylglycerols (TAGs), were analyzed to examine diabetes-related lipid species and responses to treadmill exercise. Most lysophospholipid levels increased with diabetes. While DAG levels (10 from the gastrocnemius and 13 from the soleus) were >3-fold higher in diabetic rats, levels of most of these decreased after exercise in soleus but not in gastrocnemius. Levels of 5 highly abundant TAGs (52:1 and 54:3 in the gastrocnemius and 48:2, 50:2, and 52:4 in the soleus) displaying 2-fold increases in diabetic rats decreased after exercise in the soleus but not in the gastrocnemius in most cases. Thus, aerobic exercise has a stronger influence on lipid levels in the soleus than in the gastrocnemius in type 2 diabetic rats.

Phosphoproteomic analysis of the mouse brain mu-opioid (MOP) receptor

Many in vitro data have shown that the efficacy of several opioid drugs is correlated with differential mu-opioid (MOP) receptor phosphorylation. Label-free semiquantitative on-line nanoflow liquid chromatography–tandem mass spectrometry (nanoLC–MS/MS) analyses were performed to compare the endogenous MOP receptor phosphorylation patterns of mice administered with morphine, etonitazene and fentanyl. The analysis identified S363, T370 and S375 as phosphorylated residues in the carboxy-terminus. Only T370 and S375 were regulated by agonists, with a higher propensity to promote double phosphorylation for high efficacy agonists. Our study provides confirmation that differential agonist-driven multi-site phosphorylation of MOP receptor occurs in vivo and validate the use of MS to study endogenous GPCR phosphorylation.

Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m(5)C), 2'-O-methylcytidine (Cm), N(6)-methyladenosine (m(6)A), and 2'-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m(5)C, Cm and Am are tissue-specific. In addition, the 2'-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m(5)C and m(6)A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m(5)C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m(6)A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.

Lipidomic profiling of plasma and urine from patients with Gaucher disease during enzyme replacement therapy by nanoflow liquid chromatography–tandem mass spectrometry

Gaucher disease (GD) is a rare genetic disorder that arises from lipid species, especially monohexosylceramide (MHC), accumulating in different organs. GD results from a β-glucocerebrosidase deficiency, causing metabolic or neurologic complications. This study comprehensively profiled lipids from patients and healthy controls to discover active lipid species related to GD. Most studies have evaluated lipids from one type of biological sample, such as plasma, urine, or spinal fluid, which are the main sources of lipids in human bodies. The purpose of this study, however, was to collect and assess both plasma and urine samples from a group of individuals, explore the lipids, and select characteristic species that show significant differences between controls and patients from the two sources. Also, the response of lipids to enzyme replacement therapy (ERT), which is targeted to reduce excessive lipid accumulation within lysosomes, was investigated by obtaining plasma and urine from patients after receiving the therapy. Most lipid species were found in both plasma and urine but their concentrations differed, and some species were found in either plasma or urine only. Out of 125 plasma and 105 urinary lipids that were identified by nLC–ESI–MS/MS, 20 plasma and 10 urinary lipids were selected as characteristic species for having average concentrations that were significantly increased or decreased in patients by greater than 2-fold. Moreover, the concentrations of most lipids that showed greater than 2-fold of difference in patients decreased after ERT indicating that these species were directly or indirectly affected by the therapy.

Profiling of oxidized phospholipids in lipoproteins from patients with coronary artery disease by hollow fiber flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.

Oxidized phospholipids (Ox-PLs) are oxidatively modified PLs that are produced during the oxidation of lipoproteins; oxidation of low density lipoproteins especially is known to be associated with the development of coronary artery disease (CAD). In this study, different lipoprotein classes (high density, low density, and very low density lipoproteins) from pooled plasma of CAD patients and pooled plasma from healthy controls were size-sorted on a semipreparative scale by multiplexed hollow fiber flow field-flow fractionation (MxHF5), and Ox-PLs that were extracted from each lipoprotein fraction were quantified by nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). The present study showed that oxidation of lipoproteins occurred throughout all classes of lipoproteins with more Ox-PLs identified from CAD patient lipoproteins: molecular structures of 283 unique PL species (including 123 Ox-PLs) from controls and 315 (including 169 Ox-PLs) from patients were identified by data-dependent collision-induced dissociation experiments. It was shown that oxidation of PLs occurred primarily with hydroxylation of PL; in particular, a saturated acyl chain such as 16:0, 18:0, or even 18:1 at the sn-1 location of the glycerol backbone along with sn-2 acyl chains with at least two double bonds were identified. The acyl chain combinations commonly found for hydroxylated Ox-PLs in the lipoproteins of CAD patients were 16:0/18:2, 16:0/20:4, 18:0/18:2, and 18:0/20:4.

Huntingtin is associated with cytomatrix proteins at the presynaptic terminal.

Huntington's disease (HD) is a single gene disorder produced by expansion of the gene encoding huntingtin (htt), a large protein with features of a multi-functional scaffold. Expansion of htt's polyglutamine domain induces novel, toxic interactions and likely also disrupts normal htt function. Because of its predicted role as a scaffold, pursuit of huntingtin function and HD pathogenesis has focused on identifying htt-interacting proteins. Here we present a focused screen designed to identify htt-interacting proteins in the presynaptic terminal. To identify interactions that occur in situ, synaptosomes (isolated nerve terminals) from cerebral cortices, striata and hippocampi were subjected to chemical crosslinking followed by denaturation, immunoprecipitation using an anti-htt antibody, and nano-flow liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) analyses. The presynaptic cytomatrix proteins Bassoon, Piccolo/Aczonin and Ahnak were among the most consistently identified binding partners. Co-immunoprecipitation and co-fractionation studies support the conclusion that huntingtin is a component of the presynaptic cytomatrix, a complicated network of proteins that regulates the positioning and priming of synaptic vesicles. These findings implicate htt in presynaptic functioning, and suggest that aberrant organization of presynaptic components may contribute to the neurological pathology associated with HD.

Discovery of Novel Disease-specific and Membrane-associated Candidate Markers in a Mouse Model of Multiple Sclerosis

Multiple sclerosis is a chronic demyelinating disorder characterized by the infiltration of auto-reactive immune cells from the periphery into the central nervous system resulting in axonal injury and neuronal cell death. Experimental autoimmune encephalomyelitis represents the best characterized animal model as common clinical, histological, and immunological features are recapitulated. A label-free mass spectrometric proteomics approach was used to detect differences in protein abundance within specific fractions of disease-affected tissues including the soluble lysate derived from the spinal cord and membrane protein-enriched peripheral blood mononuclear cells. Tissues were harvested from actively induced experimental autoimmune encephalomyelitis mice and sham-induced ("vehicle" control) counterparts at the disease peak followed by subsequent analysis by nanoflow liquid chromatography tandem mass spectrometry. Relative protein quantitation was performed using both intensity- and fragmentation-based approaches. After statistical evaluation of the data, over 500 and 250 differentially abundant proteins were identified in the spinal cord and peripheral blood mononuclear cell data sets, respectively. More than half of these observations have not previously been linked to the disease. The biological significance of all candidate disease markers has been elucidated through rigorous literature searches, pathway analysis, and validation studies. Results from comprehensive targeted mass spectrometry analyses have confirmed the differential abundance of ∼ 200 candidate markers (≥ twofold dysregulated expression) at a 70% success rate. This study is, to our knowledge, the first to examine the cell-surface proteome of peripheral blood mononuclear cells in experimental autoimmune encephalomyelitis. These data provide a unique mechanistic insight into the dynamics of peripheral immune cell infiltration into CNS-privileged sites at a molecular level and has identified several candidate markers, which represent promising targets for future multiple sclerosis therapies. The mass spectrometry proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000255.

Qualitative and Quantitative Characterization of Plasma Proteins When Incorporating Traveling Wave Ion Mobility into a Liquid Chromatography–Mass Spectrometry Workflow for Biomarker Discovery: Use of Product Ion Quantitation As an Alternative Data Analysis Tool for Label Free Quantitation

Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography–mass spectrometry (LC–MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended.

Characterization of oxidized phospholipids in oxidatively modified low density lipoproteins by nanoflow liquid chromatography–tandem mass spectrometry

Oxidized low density lipoproteins (Ox-LDLs) have an important role in the development of age-related vascular disease, such as atherosclerosis. Ox-LDLs are defined as oxidatively modified LDLs in the blood by enzymatic or non-enzymatic oxidation of phospholipids (PLs). For the characterization of Ox-LDLs at molecular level, oxidation patterns of oxidized PL (Ox-PL) products were systematically examined with standard PL molecules (16:0/22:6-PC, 18:0/22:6-PA, and 18:0/22:6-PG), by the formation of bilayer vesicles of each standard, followed by oxidation of PL vesicles using a Cu2+ solution. This oxidative modification was applied to LDL standard materials. Nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC–ESI–MS/MS) analysis of the extracted PL mixtures resulted in the identification of 276 PLs both in the modified and non-modified LDL, including 139 Ox-PL species. Examination of the identified PL species from the standard LDL before and after oxidation supported the postulate that the specific location of an acyl chain of LPL can be exchanged between the sn-1 and -2 positions. This exchange occurs when the neighboring acyl chain is cleaved during oxidation and the polar head group of PL molecules can be dissociated to form PA molecules that result in the formation of various Ox-PA products. This study demonstrates that nLC–ESI–MSn can be utilized for the separation and structural characterization of complicated Ox-PL mixtures, including long chain products with hydroxylation or hydroperoxylation at an unsaturated acyl chain, and short chain products from the cleavage of unsaturated fatty acyl chains to form lysophospholipids (LPLs) or the truncation of an acyl chain into a shorter chain terminated with aldehyde or carboxylic acid.