Analysis of Intracellular Nucleotides by Capillary Electrophoresis–Mass Spectrometry

A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism—AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.     

Quantitative Analysis of β-Amyloid Peptides Expressed in Human Cerebrospinal Fluid by an Improved Method of Antibody-Assisted Time-of-Flight Mass Spectrometry

Establishment of diagnostic measures for early stage Alzheimer’s disease (AD) and mild cognitive impairment (MCI) is of crucial importance. Using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS), antibody-assisted MS of cerebrospinal fluid (CSF) has enabled quantitative analysis of the ratio of β-amyloid (Aβ) peptides, Aβ1-42/Aβ1-40, which has a diagnostic value for AD/MCI. To apply the MS analysis to a far wider range of CSF samples, we have established a method to analyze Aβ peptides expressed in 100 μl CSF samples quantitatively. Pretreatment of CSF samples by limit-filtration to condense peptides, and modified washing procedure using urea as denaturant, Aβ peptides of interest can be assessed with higher sensitivity by five to tenfolds to the original method. This improvement enables quantitative analysis of Aβ species from a residual amount of CSF samples, which will be occasionally obtained in case of lumbar anesthesia prior to operation and spinal tap performed for routine diagnostic purposes. Prevalence of the new procedure as laboratory test, especially among the elderly consulting for neurological clinic, will enhance the number of subjects diagnosed at early stage of AD/MCI.     

Synthesis and Mass Spectrometry Analysis of Oligonucleotides Bearing 5-Formyl-2′-Deoxyurldlne in Their Structure

Abstract

Two oligonucleotides containing FdU (1) have been synthesized. The use of the “Pac-amidites” for the natural nucleosides has allowed the incorporation of the oxidized thymine residue without protection of the aldehydic function. The oligonucleotide composition was confirmed by enzymatic digestion and electrospray mass spectrometry.     

MetaGO: Predicting Gene Ontology of Non-homologous Proteins Through Low-Resolution Protein Structure Prediction and Protein–Protein Network Mapping

Homology-based transferal remains the major approach to computational protein function annotations, but it becomes increasingly unreliable when the sequence identity between query and template decreases below 30%. We propose a novel pipeline, MetaGO, to deduce Gene Ontology attributes of proteins by combining sequence homology-based annotation with low-resolution structure prediction and comparison, and partner's homology-based protein–protein network mapping. The pipeline was tested on a large-scale set of 1000 non-redundant proteins from the CAFA3 experiment. Under the stringent benchmark conditions where templates with > 30% sequence identity to the query are excluded, MetaGO achieves average F-measures of 0.487, 0.408, and 0.598, for Molecular Function, Biological Process, and Cellular Component, respectively, which are significantly higher than those achieved by other state-of-the-art function annotations methods. Detailed data analysis shows that the major advantage of the MetaGO lies in the new functional homolog detections from partner's homology-based network mapping and structure-based local and global structure alignments, the confidence scores of which can be optimally combined through logistic regression. These data demonstrate the power of using a hybrid model incorporating protein structure and interaction networks to deduce new functional insights beyond traditional sequence homology-based referrals, especially for proteins that lack homologous function templates. The MetaGO pipeline is available at http://zhanglab.ccmb.med.umich.edu/MetaGO/.     

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization.     

Protein Docking by the Interface Structure Similarity: How Much Structure Is Needed?

The increasing availability of co-crystallized protein-protein complexes provides an opportunity to use template-based modeling for protein-protein docking. Structure alignment techniques are useful in detection of remote target-template similarities. The size of the structure involved in the alignment is important for the success in modeling. This paper describes a systematic large-scale study to find the optimal definition/size of the interfaces for the structure alignment-based docking applications. The results showed that structural areas corresponding to the cutoff values <12 Å across the interface inadequately represent structural details of the interfaces. With the increase of the cutoff beyond 12 Å, the success rate for the benchmark set of 99 protein complexes, did not increase significantly for higher accuracy models, and decreased for lower-accuracy models. The 12 Å cutoff was optimal in our interface alignment-based docking, and a likely best choice for the large-scale (e.g., on the scale of the entire genome) applications to protein interaction networks. The results provide guidelines for the docking approaches, including high-throughput applications to modeled structures.     

Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract

We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.     

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.Post-translational modifications (PTMs)¹ are a rapidly expanding and important class of protein feature that broaden the functional diversity of proteins in a proteome. By definition, PTMs change protein structure and therefore have the potential to affect protein function by altering protein interactions, protein stability or catalytic activity (1, 2). As they have been found to occur on nearly every protein in the eukaryotic proteome, PTMs broadly impact nearly all known cellular processes. Over 300 different types of PTM are known, ranging from single atom modifications (e.g. oxide) to small protein modifiers (e.g. ubiquitin), which can occur on all but five amino acid residues resulting from enzymatic or nonenzymatic processes (3). Over 220,000 distinct PTM sites have been experimentally identified across ∼77,000 different proteins to date (dbPTM; http://dbptm.mbc.nctu.edu.tw/statistics.php) – numbers that continue to grow exponentially because of improved methods for high throughput detection by mass spectrometry (MS). By virtue of how they are detected, most PTM data are sequence-linked and lack structural context.The function of most PTMs is unknown because the rate of PTM detection far surpasses the rate at which any one modification can be studied empirically. Moreover, the functional impact of every PTM is likely not equivalent (4). For example, computational analysis of phosphorylation sites in yeast and human proteomes indicate that well-conserved phosphosites are more likely to have a functional consequence compared with poorly conserved sites, yet only a fraction of phosphosites are well conserved (5, 6). Consequently, the development of tools that provide functional prioritization of PTMs could have a broad impact on our understanding of protein regulation, biological mechanism, and molecular evolution.The emerging need for methods that predict the functional impact of a PTM has not yet been met. Longstanding methods capitalize predominantly on the sequence context of PTMs and have been used to predict sites of modification (expasy.org/proteomics/post-translational_modification) and to compare enzyme/substrate interactions (7–9). More recently, studies aimed at expanding the parameters associated with functional PTMs have emerged. In these cases, a set of common features correlated with functional importance are derived from the analysis of PTMs within and between organisms including: number of PTM observations at a multiple sequence alignment position (i.e. hotspots), measures of co-occurrence between different PTMs (e.g. distance between phosphorylation and ubiquitination sites), biological dynamics (up or down-regulation), and protein–protein interaction influence (7, 10–12). Recent efforts to provide structural context by linking individual PTMs to three-dimensional structures in the protein data bank (PDB) have also been described (13, 14). However, these resources are extensions of existing PTM databases that allow visualization of single instances of modification onto individual proteins, but do not provide quantitative or analytical value.In principle, combining PTM hotspot and structural analysis would offer multiple advantages over any one approach used in isolation. Sequence homology provides protein family membership—thereby clustering PTMs into hotspots for groups of proteins to provide information about: (1) the evolutionary conservation and (2) observation frequencies of PTMs within the family. A primary consequence of their sequence homology is that members of a protein family will exhibit similar structures and protein interactions—features that dictate the function of protein systems. A secondary consequence is that PTM hotspots generated by alignment can be projected onto family-representative protein structures, which places each PTM hotspot into a three-dimensional context that can be visualized for each family. The structural context enabled by this projection can also provide spatial information about the PTM site that can supplement the sequence characteristics of the hotspot, namely: (3) solvent accessibility, which provides an estimate of whether a modification could occur on the folded protein; and (4) protein interface residence, which indicates the potential of the PTM to disrupt protein–protein interactions. Despite the theoretical advantages, no single tool has been developed that exploits the quantitative output from both sequence and structural data to evaluate the function potential of PTMs.Here we describe a new analytical method – Structural Analysis of PTM Hotspots (SAPH-ire), which ranks PTM hotspots by their potential to impact biological function for distinct protein families (Fig. 1). We demonstrate the application of SAPH-ire to the complete set of PTMs for eight distinct protein families including large heterotrimeric G proteins—revealing high-ranking hotspots for which a biological function has not yet been determined. In particular, SAPH-ire revealed the N-terminal tail (Nt) of G protein gamma (Gγ) subunits as one of the highest ranking PTM hotspots for heterotrimeric G proteins (Gα, Gβ, and Gγ). We tested this prediction by monitoring the phosphorylation state and mutation effects of phosphorylation sites in the N terminus of the yeast Gγ subunit (Ste18). Consistent with SAPH-ire predictions, we found that phosphorylation of Ste18-Nt is biologically responsive to a GPCR stimulus and that phospho-null or phospho-mimic mutation of these sites controls protein abundance in an opposite manner in vivo. Thus, SAPH-ire is a powerful new method for predicting the function potential of PTM hotspots, which can guide empirical research toward the discovery of new protein regulatory elements based on high-throughput proteomics.Open in a separate windowFig. 1.Schematic diagram of the SAPH-ire method. A, SAPH-ire integrates InterPro, the Protein Data bank (PDB) and a customized database of experimentally validated PTMs. Uniprot entries with PTMs that belong to specific InterPro-classified protein families undergo multiple-sequence alignment (MSA) and PTM hotspot analysis (HSA), which layers all PTMs for a given alignment position in the MSA. The total PTMs observed in each hotspot and the conservation of a modifiable residue (e.g. conservation lysine at a ubiquitination hotspot) at the hotspot are quantified. B, PTM hotspots within the protein family are then projected onto all known crystal structures for the family using the Structural Projection of PTMs (SPoP) tool. From the structural topology of PTM hotspots generated by SPoP, the solvent accessible surface area (SASA) and protein interface residence is quantified for each hotspot. C, PTM Function Potential Calculator (FPC) integrates the output from HSA and SPoP, resulting in PTM function potential scores for each hotspot. The function potential score can be used to rank PTM hotspots within or between protein families – prioritizing hotspots with the greatest potential to be biologically regulated and/or effect a biological function for the protein family of interest.     

Colloidal Silver Staining of Electroblotted Proteins for High Sensitivity Peptide Mapping by Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry.     

Mitochondrial Protein Import: A Matter of Death?

Mitochondria play a central role not only in energy generation but also for apoptosis. A key step in mitochondrial apoptosis is the release of mitochondrial proteins, most importantly cytochrome c. This release is orchestrated by the pro- and anti-apoptotic members of the Bcl-2 protein family. The functions of these Bcl-2 family members are clear in terms of order and of principle: the pro-apoptotic BH3-only protein group contains the triggers, which cause the activation of the effectors Bax and Bak, while the anti-apoptotic Bcl-2-like proteins prevent this activation. However, the molecular details are still insufficiently clear and the proposed models have certain gaps and are partly contradictory. We have recently presented evidence that targeting to mitochondria of at least one BH3-only protein is essential for its pro-apoptotic functions. Here we discuss how this mechanism might fit into and expand existing models and speculate about the potential implications of this finding.     

Structure of Signal-regulatory Protein ��: A LINK TO ANTIGEN RECEPTOR EVOLUTION*

Signal-regulatory protein α (SIRPα) is a myeloid membrane receptor that interacts with the membrane protein CD47, a marker of self. We have solved the structure of the complete extracellular portion of SIRPα, comprising three immunoglobulin superfamily domains, by x-ray crystallography to 2.5 Å resolution. These data, together with previous data on the N-terminal domain and its ligand CD47 (possessing a single immunoglobulin superfamily domain), show that the CD47-SIRPα interaction will span a distance of around 14 nm between interacting cells, comparable with that of an immunological synapse. The N-terminal (V-set) domain mediates binding to CD47, and the two others are found to be constant (C1-set) domains. C1-set domains are restricted to proteins involved in vertebrate antigen recognition: T cell antigen receptors, immunoglobulins, major histocompatibility complex antigens, tapasin, and β2-microglobulin. The domains of SIRPα (domains 2 and 3) are structurally more similar to C1-set domains than any cell surface protein not involved in antigen recognition. This strengthens the suggestion from sequence analysis that SIRP is evolutionarily closely related to antigen recognition proteins.Signal-regulatory protein α (SIRPα)⁴ is a membrane receptor present on myeloid cells and neurons that interacts with the widely distributed cell surface protein CD47 (reviewed in Refs. 1 and 2). Absence of CD47 leads to uptake of cells via macrophages, indicating that CD47 acts as a marker of self (3). SIRPα gives inhibitory signals through immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic region that interact with phosphatases SHP-1 and SHP-2 (4). Binding of the N-terminal immunoglobulin superfamily (IgSF) V-set domain of SIRPα (SIRPα d1) to the single IgSF domain of CD47 is mediated by the loops of the SIRPα IgSF domain, analogous to the interactions mediated by antigen receptors, albeit involving only a single domain (5, 6). This type of binding distinguishes the CD47-SIRPα interaction from that of many interactions at the cell surface involving IgSF domains such as CD2-CD58, where the face of the IgSF domain is involved (7). SIRPα domains 2 and 3 (d2 and d3) show amino acid sequence similarity to IgSF C1-set domains (8). Since IgSF C1-set domains have only been confirmed in vertebrate antigen receptors and associated proteins (Ig light and heavy chains, T cell receptor chains, MHC class I and II and related proteins, β2-microglobulin, and very recently tapasin (9)) of the vertebrate adaptive immune system, it was suggested that SIRPα might have evolved from a precursor of the antigen receptors (8).We describe here the crystal structure of the full three-domain extracellular region of SIRPα, revealing that the topology of the CD47-SIRPα interaction is compatible with productive engagement occurring when cells come together in synapse-like contacts. We show that the two membrane-proximal IgSF domains are particularly close in structure to C1-set IgSF domains. This, together with the presence of an IgSF V-set domain mediating ligand recognition, suggests that SIRPα is related to a key precursor in the evolution of vertebrate antigen receptors.     

AMP-Activated Protein Kinase: A Universal Regulator of Autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests that AMPK inhibits mTORC1 and autophagy also in non-starved cells. Various Ca²⁺ mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate AMPK via activation of Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β), and this pathway is required for Ca²⁺-induced mTORC1 inhibition and autophagy. Thus, we propose that an increase in free cytosolic Ca²⁺ ([Ca²⁺]c) induces autophagy via the CaMKK/β-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.

Addendum to:

Control of Macroautophagy by Calcium, Calmodulin-Dependent Kinase Kinase-β and Bcl-2

M. Høyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen and M. Jäättelä

Mol Cell 2007; 25:193-205     

Characterization of Glyoxalase I (E. coli)—Inhibitor Interactions by Electrospray Time-of-Flight Mass Spectrometry and Enzyme Kinetic Analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Quantitative Analysis of Protein and Gene Expression in Salivary Glands of Sjogren’s-Like Disease NOD Mice Treated by Bone Marrow Soup

Background

Bone marrow cell extract (termed as BM Soup) has been demonstrated to repair irradiated salivary glands (SGs) and restore saliva secretion in our previous study. In the present study, we aim to investigate if the function of damaged SGs in non-obese diabetic (NOD) mice can be restored by BM Soup treatment and the molecular alterations associated with the treatment.

Methods

Whole BM cells were lysed and soluble intracellular contents (“BM Soup”) were injected I.V. into NOD mice. Tandem mass tagging with 2-D liquid chromatography-mass spectrometry was used to quantify proteins in the submandibular glands (SMGs) between untreated and BM Soup-treated mice. Quantitative PCR was used to identify genes with altered expression in the treated mice.

Results BM Soup

restored salivary flow rates to normal levels and significantly reduced the focus scores of SMGs in NOD mice. More than 1800 proteins in SMG cells were quantified by the proteomic approach. Many SMG proteins involved in inflammation and apoptosis were found to be down-regulated whereas those involved in salivary gland biology and development/regeneration were up-regulated in the BM Soup-treated mice. qPCR analysis also revealed expression changes of growth factors and cytokines in the SMGs of the treated NOD mice.

Conclusion

BM Soup treatment is effective to restore the function of damaged SGs in NOD mice. Through gene/protein expression analysis, we have found that BM Soup treatment might effectuate via inhibiting apoptosis, focal adhesion and inflammation whereas promoting development, regeneration and differentiation of the SG cells in NOD mice. These findings provide important insights on the potential mechanisms underlying the BM Soup treatment for functional restoration of damaged SGs in NOD mice. Additional studies are needed to further confirm the identified target genes and their related signaling pathways that are responsible for the BM Soup treatment.     

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu

The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1O^γ, Asp17O^δ1 and Lys32N^ε, it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH₂ to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified.