Effectiveness of Behavioral and Pharmacologic Interventions for Depressive Symptoms After Spinal Cord Injury: Findings From a Systematic Review.

OBJECTIVE: To summarize and evaluate evidence regarding the efficacy of interventions for depressive symptoms in adults living with spinal cord injury (SCI) and comorbid major depressive disorder or significant depressive symptoms to inform the development of clinical practice guidelines. DATA SOURCES: Articles published since 2013 and available in Medline, The Cochrane Library, Embase, Scopus, CINAHL, or PsycINFO. Databases were searched in June 2022 and updated November 2023. STUDY SELECTION: Inclusion criteria: age 18 years or older, traumatic SCI, and clinically significant depression (Population), mental health interventions including behavioral, pharmacologic, and complementary and alternative medicine (Intervention), inclusion of a control group (Comparator), with a primary outcome of depression symptom reduction (Outcome). Criteria were applied by multiple reviewers and disagreements were reconciled via unanimous decision among the entire research team. Eight articles of 2780 screened met the selection criteria. DATA EXTRACTION: Data were extracted independently by multiple reviewers. Two reviewers independently assigned a quality score using the guidelines described by Hawker and associates and independently evaluated the risk of bias of each article using version 2 of the Cochrane risk-of-bias tool. DATA SYNTHESIS: All studies assessed depressive symptoms during participant recruitment, screening, and/or at a baseline assessment stage. Pharmacotherapy with venlafaxine XR and several behavioral interventions appear promising, including an online mindfulness course and eye movement desensitization and reprocessing therapy. Remote interventions may be effective in reaching individuals who are unable to travel to in-person therapy sessions. CONCLUSIONS: This systematic review provides valuable information for clinicians who treat individuals with SCI and comorbid major depressive disorder or significant depressive symptoms. It highlights the importance of considering a variety of interventions and individualizing treatment to meet individuals' needs and preferences. Future research should aim to identify effective interventions for treating depressive symptoms in individuals with SCI and optimal delivery methods for these interventions.

The Development of an Innovative Crowdsourced Resident Procedure Team Model to Improve Bedside Procedural Proficiency in the Inpatient Setting.

Background Bedside procedures are a necessary skill for many residents. Practice changes, including the discontinuation of a minimum number of procedures required by the American Board of Internal Medicine, may have resulted in decreased incentive for residents to seek procedural opportunities. Objective To improve residents' procedural output and confidence in abdominal paracentesis, arterial and central venous line placement, nasogastric intubation, and ultrasound-guided peripheral intravenous catheter insertions (USPIV). Methods A novel Resident Procedure Team (RPT) model was created using crowdsourced proficient (having completed ≥5 procedures) near-peers in combination with peer-led USPIV simulation workshops to increase the number of supervising residents available. Procedure logs and the number of residents who became qualified to perform and supervise procedures were tracked from July 2018 to June 2022 and compared before and after the implementation of the RPT in July 2020. Results Implementing the novel RPT model significantly increased the number of procedures performed (1875 procedures post-RPT vs 1292 pre-RPT; P=.02). Abdominal paracentesis increased from 411 to 482 (17.3%), central venous line placement increased from 344 to 401 (16.6%), USPIV increased from 318 to 389 (22.3%), arterial line placement increased from 189 to 360 (90.5%), and nasogastric intubation increased from 30 to 243 (710.0%). Resident confidence levels increased significantly after RPT-led USPIV workshops (P<.05 for all). Conclusions Implementation of a novel, crowdsourced, resident-led procedure team and peer-led USPIV workshops helped increase the number of procedures performed by residents.

Kinetics of Human Mutant Tau Prion Formation in the Brains of 2 Transgenic Mouse Lines.

Importance: Accumulation of the protein tau is a defining characteristic of several neurodegenerative diseases. Thorough assessment of transgenic (Tg) mouse lines that replicate this process is critical for establishing the models used for testing anti-tau therapeutics in vivo. Objective: To define a consistent mouse model of disease for use in future compound efficacy studies. Design, Setting, and Participants: In this time course study, cohorts of Tg and control mice were euthanized at defined intervals. Collected brains were bisected down the midline. One half was frozen and used to measure the tau prion content, while the other half was fixed for immunostaining with anti-tau antibodies. All mice were maintained at the Hunters Point Animal Facility at the University of California, San Francisco, and all experiments were performed at the Mission Bay Campus of the University of California, San Francisco. Study animals were PS19, homozygous and hemizygous Tg(MAPT*P301S), and B6/J mice. The study dates were August 9, 2010, to October 3, 2016. Main Outcomes and Measures: Tau prions were measured using a cell-based assay. Neuropathology was measured by determining the percentage area positive for immunostaining in defined brain regions. A separate cohort of mice was aged until each mouse developed neurological signs as determined by trained animal technicians to assess mortality. Results: A total of 1035 mice were used in this time course study. These included PS19 mice (51.2% [126 of 246] male and 48.8% [120 of 246] female), Tg(MAPT*P301S+/+) mice (52.3% [216 of 413] male, 43.8% [181 of 413] female, and 3.9% [16 of 413] undetermined), Tg(MAPT*P301S+/-) mice (51.8% [101 of 195] male and 48.2% [94 of 195] female), and B6/J mice (49.7% [90 of 181] male and 50.3% [91 of 181] female). While considerable interanimal variability in neuropathology, disease onset, and tau prion formation in the PS19 mice was observed, all 3 measures of disease were more uniform in the Tg(MAPT*P301S+/+) mice. Comparing tau prion formation in Tg(MAPT*P301S+/+) mice with B6/J controls, the 95% CIs for the 2 mouse lines diverged before age 5 weeks, and significant (P < .05) neuropathology in the hindbrain of 24-week-old mice was quantifiable. Conclusions and Relevance: The assessment of disease progression using 3 criteria showed that disease onset in PS19 mice is too variable to obtain reliable measurements for drug discovery research. However, the reproducibility of tau prion formation in young Tg(MAPT*P301S+/+) mice establishes a rapid assay for compound efficacy in vivo.

Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns.

PURPOSE: To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain. EXPERIMENTAL DESIGN: Parietal cortex from individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high resolution. RESULTS: Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs, which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2'-3'-cyclic nucleotide-3' phosphodiesterase have relatively higher phosphorylation levels during HIV infection. CONCLUSIONS AND CLINICAL RELEVANCE: This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders.

Application of quantitative proteomics to the integrated analysis of the ubiquitylated and global proteomes of xenograft tumor tissues.

BACKGROUND: Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues. RESULTS: In this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level. CONCLUSIONS: This is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.

Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes.

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 (small ubiquitin-related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods, we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere- and kinetochore-associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs.

Identification of putative biomarkers for HIV-associated neurocognitive impairment in the CSF of HIV-infected patients under cART therapy determined by mass spectrometry.

We identified and measured proteins in the cerebral spinal fluid (CSF) involved in HIV-associated neurological disorders. Protein levels were determined by mass spectrometry (MS) in pooled CSF taken from three patient groups (human immunodeficiency virus (HIV)-1-infected patients that developed HIV-associated neurocognitive disorders (HANDs), HIV-1-infected patients without HAND, and healthy controls). Pools were generated from 10 patients each per group. CSF from individual patient groups were digested with trypsin and separately labeled using with isobaric tags for relative and absolute quantitation (iTRAQ). After combining all samples in one, peptides were extensively fractionated by offline two-dimensional separation and identified by tandem MS. One hundred and ninety three proteins were deemed to be interpretable for quantitation based on permutation tests with a 95 % confidence interval with a p value ≤ 0.05. Using a cutoff of 1.5-fold for upregulation and 0.6 for downregulation, 16 proteins were differentially expressed in HIV + HAND (reporter p value ≤0.05) with seven of them previously described as HIV-interacting proteins: endoplasmin, mitochondrial damage mediator-BH3-interacting domanin death agonist, orosomucoid, apolipoprotein E, metalloproteinase inhibitor 2, peroxiredoxin-2, and the nuclear protein, ruvB-like 2. Several previously unidentified proteins with possible neurological implication in HIV patients include forming-binding protein 1, C-reactive protein, leukocyte-associated immunoglobulin receptor 1, renin receptor, mediator of RNA polymerase II transcription subunit 14, multimerin-2, alpha-N-acetylglucosaminidase, caldesmon, and cadherin EGF LAG G-type receptor. Our results suggest that not only a few but possibly a combination of biomarkers that are highly correlated can predict neurocognitive status in HIV-infected patients and might be involved in monocyte or macrophage activation.

Identification of nitrated immunoglobulin variable regions in the HIV-infected human brain: implications in HIV infection and immune response.

HIV can infiltrate the brain and lead to HIV-associated neurocognitive disorders (HAND). The pathophysiology of HAND is poorly understood, and there are no diagnostic biomarkers for it. Previously, an increase in inducible nitric oxide synthase levels and protein tyrosine nitration in the brain were found to correlate with the severity of HAND.1,2 In this study, we analyzed human brains from individuals who had HIV infection without encephalitis and with encephalitis/HAND and compared them to the brains of healthy individuals. We identified the nitrated proteins and determined the sites of modification using affinity enrichment followed by high-resolution and high-mass-accuracy nanoLC-MS/MS. We found that nitrated proteins were predominantly present in the HIV-infected individuals with encephalitis, and, interestingly, the modifications were predominantly located on immunoglobulin variable regions. Our molecular model indicated potential interactions with HIV envelope proteins and changes on the heavy and light chain interface upon the nitration and nitrohydroxylation of these residues. Therefore, our findings suggest a role for these modifications in the immune response, which may have implications in disease pathogenesis.

Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a "two-active site" model for multiple histone H3 lysine 4 methylation.

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a "two-active site" model for multiple H3K4 methylation by the MLL1 core complex.

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants.

The core histones, H2A, H2B, H3 and H4, undergo post-translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono-, di- and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high-performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom-up liquid chromatography-mass spectrometric analysis. The deuteroacetylation of unmodified or mono-methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification 'cross-talk' by correlating different PTMs on the same histone tail.

Using glycinylation, a chemical derivatization technique, for the quantitation of ubiquitinated proteins.

The quantitation of lysine post-translational modifications (PTMs) by bottom-up mass spectrometry is convoluted by the need for analogous derivatives and the production of different tryptic peptides from the unmodified and modified versions of a protein. Chemical derivatization of lysines prior to enzymatic digestion circumvents these problems and has proven to be a successful method for lysine PTM quantitation. The most notable example is the use of deuteroacetylation to quantitate lysine acetylation. In this work, levels of lysine ubiquitination were quantitated using a structurally homologous label that is chemically similar to the diglycine (GlyGly) tag, which is left at the ubiquitination site upon trypsinolysis. The LC-MS analysis of a chemically equivalent monoglycine (Gly) tag that is analogous to the corresponding GlyGly tag proved that the monoglycine tag can be used for the quantitation of ubiquitination. A glycinylation protocol was then established for the derivatization of proteins to label unmodified lysine residues with a single glycine tag. Ubiquitin multimers were used to show that after glycinylation and tryptic digestion, the mass spectrometric response from the corresponding analogous tagged peptides could be compared for relative quantitation. For a proof of principle regarding the applicability of this technique to the analysis of ubiquitination in biological samples, the glycinylation technique was used to quantitate the increase in monoubiquitinated histone H2B that is observed in yeast which lacks the enzyme responsible for deubiquitinating H2B-K123, compared to wild-type yeast.

High energy collisions on tandem time-of-flight mass spectrometers.

Long before the introduction of matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), Orbitraps, and any of the other tools that are now used ubiquitously for proteomics and metabolomics, the highest performance mass spectrometers were sector instruments, providing high resolution mass measurements by combining an electrostatic energy analyzer (E) with a high field magnet (B). In its heyday, the four sector mass spectrometer (or EBEB) was the crown jewel, providing the highest performance tandem mass spectrometry using single, high energy collisions to induce fragmentation. During a time in which quadrupole and tandem triple quadrupole instruments were also enjoying increased usage and popularity, there were, nonetheless, some clear advantages for sectors over their low collision energy counterparts. Time-of-flight (TOF) mass spectrometers are high voltage, high vacuum instruments that have much in common with sectors and have inspired the development of tandem instruments exploiting single high energy collisions. In this retrospective, we recount our own journey to produce high performance TOFs and tandem TOFs, describing the basic theory, problems, and the advantages for such instruments. An experiment testing impulse collision theory (ICT) underscores the similarities with sector mass spectrometers where this concept was first developed. Applications provide examples of more extensive fragmentation, side chain cleavages, and charge-remote fragmentation, also characteristic of high energy sector mass spectrometers. Moreover, the so-called curved-field reflectron has enabled the design of instruments that are simpler, collect and focus all of the ions, and may provide the future technology for the clinic, for tissue imaging, and the characterization of microorganisms.

Oxidative stress and the HIV-infected brain proteome.

Human immunodeficiency virus (HIV) is capable of infiltrating the brain and infecting brain cells. In the years following HIV infection, patients show signs of various levels of neurocognitive problems termed HIV-associated neurocognitive disorders (HAND). Although the introduction of highly active antiretroviral therapy (HAART) has reduced the incidence of HIV-dementia, which is the most severe form of HAND, the milder forms have become more prevalent today due to the increased life expectancy of infected individuals. Pre-HAART era markers such as HIV RNA level, CD4+ count, TNF-α, MCP-1 and M-CSF are not able to clearly distinguish mild from advanced HAND. One promising approach for new biomarker discovery is the identification and quantitation of proteins that are post-translationally modified by oxidative and nitrosative species. The occurrence of oxidative and nitrosative stress in HIV-infected brain, both through the early direct and indirect effects of viral proteins and through the later effect on mitochondrial integrity during apoptosis, is well-established. This review will focus on how the reactive species are produced in the brain after HIV infection, the specific oxidative and nitrosative species that are involved in the post-translational modification of the brain proteome, and the methods that are currently used for the detection of such modified proteins. This review also provides an overview of related research pertaining to oxidative stress-related HAND using cerebrospinal fluid and human brain tissue.

The conserved set of host proteins incorporated into HIV-1 virions suggests a common egress pathway in multiple cell types.

HIV-1 incorporates a large array of host proteins into virions. Determining the host protein composition in HIV virions has technical difficulties, including copurification of microvesicles. We developed an alternative purification technique using cholesterol that differentially modulates the density of virions and microvesicles (density modification, DM) allowing for high-yield virion purification that is essential for tandem mass spectrometric and quantitative proteomic (iTRAQ) analysis. DM purified virions were analyzed using iTRAQ and validated against Optiprep (60% iodixanol) purified virions. We were able to characterize host protein incorporation in DM-purified HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages (MDM) derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the MDM derived and T-cell derived data set. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, and a handful of other proteins, many of which have previously been documented to interact with viral proteins. Other proteins of note were ERM proteins, the dynamin domain containing protein EH4, a phosphodiesterase, and cyclophilin A. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral life cycle. Additionally, identified common set proteins are predicted to interact with >1000 related human proteins. Many of these secondary interacting proteins are reported to be incorporated into virions, including ERM proteins and adhesion molecules. Thus, only a few direct interactions between host and viral proteins may dictate the host protein composition in virions. Ultimately, interaction and expression differences in host proteins between cell types may drive virion phenotypic diversity, despite conserved viral protein-host protein interactions between cell types.

Advances in membrane proteomics and cancer biomarker discovery: current status and future perspective.

Membrane proteomic analysis has been proven to be a promising tool for identifying new and specific biomarkers that can be used for prognosis and monitoring of various cancers. Membrane proteins are of great interest particularly those with functional domains exposed to the extracellular environment. Integral membrane proteins represent about one-third of the proteins encoded by the human genome and assume a variety of key biological functions, such as cell-to-cell communication, receptor-mediated signal transduction, selective transport, and pharmacological actions. More than two-thirds of membrane proteins are drug targets, highlighting their immensely important pharmaceutical significance. Most plasma membrane proteins and proteins from other cellular membranes have several PTMs; for example, glycosylation, phosphorylation, and nitrosylation, and moreover, PTMs of proteins are known to play a key role in tumor biology. These modifications often cause change in stoichiometry and microheterogeneity in a protein molecule, which is apparent during electrophoretic separation. Furthermore, the analysis of glyco- and phosphoproteome of cell membrane presents a number of challenges mainly due to their low abundance, their large dynamic range, and the inherent hydrophobicity of membrane proteins. Under pathological conditions, PTMs, such as phosphorylation and glycosylation are frequently altered and have been recognized as a potential source for disease biomarkers. Thus, their accurate differential expression analysis, along with differential PTM analysis is of paramount importance. Here we summarize the current status of membrane-based biomarkers in various cancers, and future perspective of membrane biomarker research.

Development of protein biomarkers in cerebrospinal fluid for secondary progressive multiple sclerosis using selected reaction monitoring mass spectrometry (SRM-MS).

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS). The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease. RESULTS: Using high performance liquid chromatography-coupled mass spectrometry (HPLC); we have established a highly specific and sensitive selected reaction monitoring (SRM) assay. Our multiplexed SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 26 proteins present in cerebrospinal fluid (CSF). Protein levels in CSF were generally ~200-fold lower than that in human sera. A limit of detection (LOD) was determined to be as low as one femtomol. We processed and analysed CSF samples from a total of 22 patients with SPMS, 7 patients with SPMS treated with lamotrigine, 12 patients with non-inflammatory neurological disorders (NIND) and 10 healthy controls (HC) for the levels of these 26 selected potential protein biomarkers. Our SRM data found one protein showing significant difference between SPMS and HC, three proteins differing between SPMS and NIND, two proteins between NIND and HC, and 11 protein biomarkers showing significant difference between a lamotrigine-treated and untreated SPMS group. Principal component analysis (PCA) revealed that these 26 proteins were correlated, and could be represented by four principal components. Overall, we established an efficient platform to develop and verify protein biomarkers in CSF, which can be easily adapted to other proteins of interest related to neurodegenerative diseases. CONCLUSIONS: A highly specific and sensitive multiplex SRM-MS assay was established for development and verification of CSF protein biomarkers in SPMS. Five proteins were found to be expressed significantly differently between the three cohorts, SPMS, NIND and HC and 11 proteins associated with lamotrigine treatment, which we expect will further our current understanding of SPMS disease pathology and/or therapeutic intervention.

Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion.

Small-ubiquitin-like modifier (SUMO) is a posttranslational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization and mitosis, among other cellular processes, are known to occur. However, although the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly "tag" that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32-amino-acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches, which serve to shorten the SUMO tag and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-time-of-flight (TOF) and LC/MS instrumentation, along with collision induced dissociation (CID) and electron transfer dissociation (ETD).

Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

Robust two-dimensional separation of intact proteins for bottom-up tandem mass spectrometry of the human CSF proteome.

Cerebrospinal fluid (CSF) is produced in the brain by cells in the choroid plexus at a rate of 500 mL/day. It is the only body fluid in direct contact with the brain. Thus, any changes in the CSF composition will reflect pathological processes and make CSF a potential source of biomarkers for different disease states. Proteomics offers a comprehensive view of the proteins found in CSF. In this study, we use a recently developed nongel based method of sample preparation of CSF followed by liquid chromatography-high accuracy mass spectrometry (LC-MS) for MS and MS/MS analyses, allowing unambiguous identification of peptides/proteins. Gel-eluted liquid fraction entrapment electrophoresis (Gelfree) is used to separate a CSF complex protein mixture in 12 user-selectable liquid-phase molecular weight fractions. Using this high throughput workflow, we have been able to separate CSF intact proteins over a broad mass range (3.5-100 kDa) with high resolution (between 15 and 100 kDa) in 2 h and 40 min. We have completely eliminated albumin and were able to interrogate the low abundance CSF proteins in a highly reproducible manner from different CSF samples at the same time. Using LC-MS as a downstream analysis, we identified 368 proteins using MidiTrap G-10 desalting columns and 166 proteins (including 57 unique proteins) using Zeba spin columns with a 5% false discovery rate (FDR). Prostaglandin D2 synthase, Chromogranin A, Apolipoprotein E, Chromogranin B, Secretogranin III, Cystatin C, VGF nerve growth factor, and Cadherin 2 are a few of the proteins that were characterized. Gelfree-LC-MS is a robust method for the analysis of the human proteome that we will use to develop biomarkers for several neurodegenerative diseases and to quantitate these markers using multiple reaction monitoring.

Analysis of Histone Modifications from Tryptic Peptides of Deuteroacetylated Isoforms.

The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.