Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis.

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin).

Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated.

Metabolomic profiling of anionic metabolites in head and neck cancer cells by capillary ion chromatography with Orbitrap mass spectrometry.

A highly sensitive platform coupling capillary ion chromatography (Cap IC) with Q Exactive mass spectrometer has been developed for metabolic profiling of head and neck squamous cell carcinoma (HNSCC) cells. The Cap IC allowed an excellent separation of anionic polar metabolites, and the sensitivities increased by up to 100-fold compared to reversed-phase liquid chromatography and hydrophilic interaction chromatography performed at either high- or capillary-flow rates. The detection limits for a panel of standard metabolites were between 0.04 to 0.5 nmol/L (0.2 to 3.4 fmol) at a signal-to-noise ratio of 3. This platform was applied to an untargeted metabolomic analysis of head and neck cancer cells and stem-like cancer cells. Differential metabolomics analysis identified significant changes in energy metabolism pathways (e.g., glycolysis and tricarboxylic acid cycle). These experiments demonstrate Cap IC/MS as a powerful metabolomics tool by providing enhanced separation and sensitivity of polar metabolites combined with high resolution and accurate mass measurement (HR/AM) capabilities to differentiate isobaric metabolites.

The chromosome-centric human proteome project: a call to action.

The grand vision of the human proteome project (HPP) is moving closer to reality with the recent announcement by HUPO of the creation of the HPP consortium in charge of the development of a two-part HPP, one focused on the description of proteomes of biological samples or related to diseases (B/D-HPP) and the other dedicated to a systematic description of proteins as gene products encoded in the human genome (the C-HPP). This new initiative of HUPO seeks to identify and characterize at least one representative protein from every gene, create a protein distribution atlas and a protein pathway or network map. This vision for proteomics can be the roadmap of biological and clinical research for years to come if it delivers on its promises. The Industrial Advisory Board (IAB) to HUPO shares the visions of C-HPP. The IAB will support and critically accompany the overall project goals and the definitions of the critical milestones. The member companies are in a unique position to develop hardware and software, reagents and standards, procedures, and workflows to ensure a reliable source of tools available to the proteomics community worldwide. In collaboration with academia, the IAB member companies can and must develop the tools to reach the ambitious project goals. We offer to partner with and challenge the academic groups leading the C-HPP to define both ambitious and obtainable goals and milestones to make the C-HPP a real and trusted resource for future biology.

Charge-based interactions through peptide position 4 drive diversity of antigen presentation by human leukocyte antigen class I molecules.

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

Capillary endothelial Na(+), K(+), ATPase transporter homeostasis and a new theory for migraine pathophysiology.

BACKGROUND: Cerebrospinal fluid sodium concentration ([Na(+)](csf)) increases during migraine, but the cause of the increase is not known. OBJECTIVE: Analyze biochemical pathways that influence [Na(+)](csf) to identify mechanisms that are consistent with migraine. METHOD: We reviewed sodium physiology and biochemistry publications for links to migraine and pain. RESULTS: Increased capillary endothelial cell (CEC) Na(+), K(+), -ATPase transporter (NKAT) activity is probably the primary cause of increased [Na(+)](csf). Physiological fluctuations of all NKAT regulators in blood, many known to be involved in migraine, are monitored by receptors on the luminal wall of brain CECs; signals are then transduced to their abluminal NKATs that alter brain extracellular sodium ([Na(+)](e)) and potassium ([K(+)](e)). CONCLUSIONS: We propose a theoretical mechanism for aura and migraine when NKAT activity shifts outside normal limits: (1) CEC NKAT activity below a lower limit increases [K(+)](e), facilitates cortical spreading depression, and causes aura; (2) CEC NKAT activity above an upper limit elevates [Na(+)](e), increases neuronal excitability, and causes migraine; (3) migraine-without-aura may arise from CEC NKAT over-activity without requiring a prior decrease in activity and its consequent spreading depression; (4) migraine triggers disturb, and treatments improve, CEC NKAT homeostasis; (5) CEC NKAT-induced regulation of neural and vasomotor excitability coordinates vascular and neuronal activities, and includes occasional pathology from CEC NKAT-induced apoptosis or cerebral infarction.

The morphology and biochemistry of nanostructures provide evidence for synthesis and signaling functions in human cerebrospinal fluid.

BACKGROUND: Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission. METHODS: Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy. RESULTS: Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin. CONCLUSION: Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms via volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.

PPKs mediate direct signal transfer from phytochrome photoreceptors to transcription factor PIF3.

Upon light-induced nuclear translocation, phytochrome (phy) sensory photoreceptors interact with, and induce rapid phosphorylation and consequent ubiquitin-mediated degradation of, transcription factors, called PIFs, thereby regulating target gene expression and plant development. Nevertheless, the biochemical mechanism of phy-induced PIF phosphorylation has remained ill-defined. Here we identify a family of nuclear protein kinases, designated Photoregulatory Protein Kinases (PPK1-4; formerly called MUT9-Like Kinases (MLKs)), that interact with PIF3 and phyB in a light-induced manner in vivo. Genetic analyses demonstrate that the PPKs are collectively necessary for the normal light-induced phosphorylation and degradation of PIF3. PPK1 directly phosphorylates PIF3 in vitro, with a phosphosite pattern that strongly mimics the light-induced pattern in vivo. These data establish that the PPKs are directly involved in catalysing the photoactivated-phy-induced phosphorylation of PIF3 in vivo, and thereby are critical components of a transcriptionally centred signalling hub that pleiotropically regulates plant growth and development in response to multiple signalling pathways.

Protein analysis in human cerebrospinal fluid: Physiological aspects, current progress and future challenges.

The introduction of lumbar puncture into clinical medicine over 100 years ago marks the beginning of the study of central nervous system diseases using the human cerebrospinal fluid (CSF). Ever since, CSF has been analyzed extensively to elucidate the physiological and biochemical bases of neurological disease. The proximity of CSF to the brain makes it a good target for studying the pathophysiology of brain functions, but the barrier function of the CSF also impedes its diagnostic value. Today, measurements to determine alterations in the composition of CSF are central in the differential diagnosis of specific diseases of the central nervous system (CNS). In particular, the analysis of the CSF protein composition provides crucial information in the diagnosis of CNS diseases. This enables the assessment of the physiology of the blood-CSF barrier and of the immunology of intrathecial responses. Besides those routine measurements, protein compositional studies of CSF have been extended recently to many other proteins in the expectation that comprehensive analysis of lower abundance CSF proteins will lead to the discovery of new disease markers. Disease marker discovery by molecular profiling of the CSF tissue has the enormous potential of providing many new disease relevant molecules. New developments in protein profiling techniques hold promise for the discovery and validation of relevant disease markers. In this review, we summarize the current efforts and progress in CSF protein profiling measurements using conventional and current protein analysis tools. We also discuss necessary development in methodology in order to have the highest impact on the study of the molecular composition of CSF proteins.

Identification of disease markers in human cerebrospinal fluid using lipidomic and proteomic methods.

Lipids comprise the bulk of the dry mass of the brain. In addition to providing structural integrity to membranes, insulation to cells and acting as a source of energy, lipids can be rapidly converted to mediators of inflammation or to signaling molecules that control molecular and cellular events in the brain. The advent of soft ionization procedures such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have made it possible for compositional studies of the diverse lipid structures that are present in brain. These include phospholipids, ceramides, sphingomyelin, cerebrosides, cholesterol and their oxidized derivatives. Lipid analyses have delineated metabolic defects in disease conditions including mental retardation, Parkinson's Disease (PD), schizophrenia, Alzheimer's Disease (AD), depression, brain development, and ischemic stroke. In this review, we examine the structure of the major lipid classes in the brain, describe methods used for their characterization, and evaluate their role in neurological diseases. The potential utility of characterizing lipid markers in the brain, with specific emphasis on disease mechanisms, will be discussed. Additionally, we describe several proteomic strategies for characterizing lipid-metabolizing proteins in human cerebrospinal fluid (CSF). These proteins may be potential therapeutic targets since they transport lipids required for neuronal growth or convert lipids into molecules that control brain physiology. Combining lipidomics and proteomics will enhance existing knowledge of disease pathology and increase the likelihood of discovering specific markers and biochemical mechanisms of brain diseases.

Prostaglandin D synthase isoforms from cerebrospinal fluid vary with brain pathology.

Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS) has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls) with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson's disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

Charger: combination of signal processing and statistical learning algorithms for precursor charge-state determination from electron-transfer dissociation spectra.

Tandem mass spectrometry in combination with liquid chromatography has emerged as a powerful tool for characterization of complex protein mixtures in a high-throughput manner. One of the bioinformatics challenges posed by the mass spectral data analysis is the determination of precursor charge when unit mass resolution is used for detecting fragment ions. The charge-state information is used to filter database sequences before they are correlated to experimental data. In the absence of the accurate charge state, several charge states are assumed. This dramatically increases database search times. To address this problem, we have developed an approach for charge-state determination of peptides from their tandem mass spectra obtained in fragmentations via electron-transfer dissociation (ETD) reactions. Protein analysis by ETD is thought to enhance the range of amino acid sequences that can be analyzed by mass spectrometry-based proteomics. One example is the improved capability to characterize phosphorylated peptides. Our approach to charge-state determination uses a combination of signal processing and statistical machine learning. The signal processing employs correlation and convolution analyses to determine precursor masses and charge states of peptides. We discuss applicability of these methods to spectra of different charge states. We note that in our applications correlation analysis outperforms the convolution in determining peptide charge states. The correlation analysis is best suited for spectra with prevalence of complementary ions. It is highly specific but is dependent on quality of spectra. The linear discriminant analysis (LDA) approach uses a number of other spectral features to predict charge states. We train LDA classifier on a set of manually curated spectral data from a mixture of proteins of known identity. There are over 5000 spectra in the training set. A number of features, pertinent to spectra of peptides obtained via ETD reactions, have been used in the training. The loading coefficients of LDA indicate the relative importance of different features for charge-state determination. We have applied our model to a test data set generated from a mixture of 49 proteins. We search the spectra with and without use of the charge-state determination. The charge-state determination helps to significantly save the database search times. We discuss the cost associated with the possible misclassification of charge states.

On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modifications.

We have expanded our recent on-line LC-MS platform for large peptide analysis to combine collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10,000 Da) from complex proteins, such as beta-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approximately >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g.,

ChromAlign: A two-step algorithmic procedure for time alignment of three-dimensional LC-MS chromatographic surfaces.

We present an algorithmic approach to align three-dimensional chromatographic surfaces of LC-MS data of complex mixture samples. The approach consists of two steps. In the first step, we prealign chromatographic profiles: two-dimensional projections of chromatographic surfaces. This is accomplished by correlation analysis using fast Fourier transforms. In this step, a temporal offset that maximizes the overlap and dot product between two chromatographic profiles is determined. In the second step, the algorithm generates correlation matrix elements between full mass scans of the reference and sample chromatographic surfaces. The temporal offset from the first step indicates a range of the mass scans that are possibly correlated, then the correlation matrix is calculated only for these mass scans. The correlation matrix carries information on highly correlated scans, but it does not itself determine the scan or time alignment. Alignment is determined as a path in the correlation matrix that maximizes the sum of the correlation matrix elements. The computational complexity of the optimal path generation problem is reduced by the use of dynamic programming. The program produces time-aligned surfaces. The use of the temporal offset from the first step in the second step reduces the computation time for generating the correlation matrix and speeds up the process. The algorithm has been implemented in a program, ChromAlign, developed in C++ language for the .NET2 environment in WINDOWS XP. In this work, we demonstrate the applications of ChromAlign to alignment of LC-MS surfaces of several datasets: a mixture of known proteins, samples from digests of surface proteins of T-cells, and samples prepared from digests of cerebrospinal fluid. ChromAlign accurately aligns the LC-MS surfaces we studied. In these examples, we discuss various aspects of the alignment by ChromAlign, such as constant time axis shifts and warping of chromatographic surfaces.

Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS.

The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a Finnigan LTQ linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluC yielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications.

Cerebrospinal fluid sodium increases in migraine.

BACKGROUND: Pharmaceuticals with calcium- or sodium-channel-blocking activity have proven useful for migraine prophylaxis, and calcium channel, sodium transporter, and sodium channel gene mutations have been found in familial hemiplegic migraine. However, it is not known whether calcium or sodium homeostasis is altered in migraine. OBJECTIVE: To compare levels of sodium, calcium, potassium, and magnesium in cerebrospinal fluid (CSF) and blood plasma between migraineurs and controls. METHODS: We recruited 20 migraineurs without aura and 11 controls prospectively, and studied migraineurs in sick (MH(+)) and well (MH(-)) states. We collected lumbar CSF and venous blood plasma, quantified elements with ion-selective electrodes or colorimetry, and determined osmolality by depression of freezing point. We compared levels of Na(+), Ca(2+), K(+), and Mg among and also within subjects who were studied in both MH(+) and MH(-) states. RESULTS: Mean CSF Na(+) levels were increased by 3 mmol/L in MH(+) compared with MH(-) and by 4 mmol/L compared to controls (P < 0.005). In 4 subjects who were sampled in both MH(+) and MH(-) states, mean CSF Na(+) concentration increased by 2 mmol/L in the MH(+) state compared with the MH(-) state (P < 0.05). Simultaneous plasma Na(+) levels did not differ among the 3 clinical groups, nor did osmolality, total Ca and Ca(2+), K(+), and total Mg levels in CSF. CONCLUSIONS: Compared to both controls and the MH(-) state, CSF Na(+) concentration increased in MH(+) independently from other clinical or pharmacological fluctuations, CSF concentrations of Ca(2+), Mg, and K(+), and blood plasma Na(+) levels. These results implicate a deviation of Na(+) homeostasis in migraine. The modestly elevated extracellular Na(+) in MH(+) may cause the neural changes that underlie clinical features of migraine.

Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods.

We compared detection sensitivity and protein sequence coverage of the adenovirus type 5 proteome achievable by liquid chromatography and tandem mass spectroscopy (LC/MS/MS) using three sample preparation and clean up methods. Tryptic digestion was performed on either purified viral proteins or whole virus, and followed by shotgun sequencing using tandem mass spectrometry for peptide identification. We used a recombinant adenovirus type 5 as a test system. The methods included separation of adenoviral proteins by reversed-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS/MS. Alternatively, the purified whole virus was digested with trypsin and the peptides separated either by one-dimensional (reversed-phase) or by two-dimensional (cation exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. A total of 11 protein species were identified from 154 peptides. All of the major viral proteins were found. In addition, two minor proteins, the 23 kDa viral protease and the late L1 protein, were identified for the first time by chromatography based assays. The 23 kDa viral protease, present at only 10 copies per virus, and representing 0.2% of the protein content of the virus, was detected by the 2D LC/MS/MS analysis of the whole virus digest from a sample containing only 70 fmols of the protein. This demonstrates the high sensitivity and selectivity of the method. The 2D LC/MS/MS analysis of the whole virus digest was also able to detect all viral proteins with copy numbers at or above 10/virus particle, with broad coverage of the amino acid sequences. Coverage ranged from 2 to 54%, a majority between 20 and 35%, suggesting the possibility of using this analysis to assess the purity of the virus preparations. This broad coverage may also provide a useful approach to identify posttranslational modifications on the structural proteins of the adenovirus.