Using Ion Mobility Spectrometry–Mass Spectrometry to Decipher the Conformational and Assembly Characteristics of the Hepatitis B Capsid Protein

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.     

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano–liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N^α-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.Histones are the most intensively studied group of basic nuclear proteins and are of great importance with regard to the organization of chromatin structure and control of gene activity. They are highly conserved during evolution, binding to and condensing eukaryotic chromosomal DNA to form chromatin. The fundamental chromatin subunit is the nucleosome, in which 166 bp of DNA are wrapped around a core histone octamer and a further ∼40 bp constitute the linker between one nucleosome core and the next. The histone octamer contains two molecules of each of the core histones H2A, H2B, H3, and H4. A fifth type of histone, referred to as linker histone (H1, H5), binds to both the DNA on the outer surface of nucleosomes and the linker DNA.There are numerous microsequence variants of linker and core histones (except H4) differing only slightly in primary sequence. In rat testis, for example, six somatic H1 subtypes, designated as H1a, H1b, H1c, H1d, H1e, and H1.0, as well as germ cell specific subtypes (i.e. H1t, H1T2, and HILS1), have been identified (1–3). Under various biological conditions, all histone proteins, for both linker and core histones, are subjected to post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination, deamidation, glycosylation, and ADP-ribosylation, which have a great influence on the epigenetic control of gene expression (4–6). The multitude of histone proteins resulting from closely related sequence variants and post-translational modifications, as well as their highly basic nature combined with hydrophobic properties, provides a major analytical challenge in current proteomics research. Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7–14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g. trypsin, Lys-C, etc.) causes the formation of many short and polar peptides that poorly interact with the reverse-phase (RP)¹ material and go undetected by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). To overcome this problem, chemical derivatization such as propionylation is often applied (15, 16).Capillary electrophoresis (CE) overcomes this disadvantage; this technique allows separations based on the mass-to-charge ratio of peptides and does not utilize their hydrophobic nature as a separation principle. The methods of electrophoresis and LC and their applicability for histone analysis have been reviewed in detail by Lindner (17). CE has proven to be a remarkably powerful method for separating individual histones and their modified forms based on their different electrophoretic mobilities. Using a bare fused silica capillary and hydroxypropylmethyl cellulose (HPMC) as a buffer additive in order to avoid undesired protein adsorption, different core and linker histones and their multiply phosphorylated and acetylated forms were successfully separated via capillary zone electrophoresis (CZE) (18–22). So far, no data have been published about the identification of histone modifications by means of capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS). LC is given preference over CE because of the difficulty of achieving on-line interfacing of CE with MS that allows stable electrospray processes without compromising the quality of separation or the detection sensitivity. However, CE-MS is a promising technique with constantly increasing importance, as documented by numerous articles (23–26).Various interfaces have been constructed to improve CESI-MS coupling (27, 28). Sheathflow interfaces are the most widely used, and although the drawback of having to dilute the analyte is inherent in this kind of interface, they offer stable electrophoretic separations and allow greater versatility in the choice of background electrolyte (BGE) and the range of flow rates (29–32). Sheathless interfaces have generated interest because no sheath liquid is added, which leads to enhanced detection sensitivity (33, 34). However, they have not been used frequently because of their limited robustness and lack of well-established interfaces and routine analysis protocols. The most widely used method for establishing the terminating electrical contact is coating the outer surface of the CE capillary tip with a conductive material (35–37). Unfortunately, the lifetimes of such coatings are generally very limited, as they suffer from deterioration under the influence of the high voltages applied.A recently published concept of a sheathless interface based on a separation capillary with a porous tip acting as a nanospray emitter overcomes these disadvantages (38). The capillary tip is etched using hydrofluoric acid until the capillary wall becomes so thin and porous that an electric contact can be established. The performance of this methodology, which combines the low-flow characteristics of CE with an integrated ESI source, is described in Refs. 39–41. Applications such as the analysis of intact proteins (42), protein–protein and protein–metal complexes (43), and ribosomal protein digests from E. coli (44) have been published. Method-inherent advantages of CESI-MS are highly efficient separations, low flow rates leading to reduced ion suppression, and greater sensitivity (40). In contrast to nano-LC, no column equilibration is needed, there are no gradient effects, and the instrumentation is less maintenance-intensive.Our group recently described important features of CESI-MS and reported the comparison of this method with LC-ESI-MS for the analysis of a 5% perchloric acid extraction of rat testis consisting mainly of different histone H1 subtypes (39). The performance of both techniques was evaluated regarding analysis time, protein sequence coverage, and number and molecular mass distribution of the identified peptides. The CESI-MS method provided shorter analysis times, narrower peaks yielding high signals, and the identification of a greater number of low molecular mass range peptides than LC-ESI-MS (39).In the current study, we investigated the analysis of post-translationally modified peptides, particularly phosphopeptides, obtained from endoproteinase Arg-C digested histones from rat testis; this organ contains the whole set of somatic and germ cell specific H1 histones, as well as numerous modified core histone proteins. CESI-MS and LC-ESI-MS were compared regarding the number and type of identified modified peptides. Without any pre-separation of the perchloric acid extraction, we found numerous known and novel modification sites in linker histones. In addition, immobilized metal-affinity chromatography (IMAC) experiments were utilized to enrich phosphopeptides prior to MS analysis. CESI-MS was also used for the rapid identification of post-translational modifications (PTMs) of rat testis core histones, which were pre-fractionated via RP-HPLC and digested with Arg-C. Using core histones from butyrate-treated mouse erythroleukemia cells, we further demonstrated that our method achieves excellent separations of intact histone subtypes and their multiply modified forms and enables the detection of the extent of PTMs in a fast and reproducible way. Our work represents the first detailed characterization of modified linker and core histone peptides and clearly demonstrates that CESI-MS is a promising alternative tool for epigenetic studies.     

Chemical Crosslinking Mass Spectrometry Reveals the Conformational Landscape of the Activation Helix of PPARγ; a Model for Ligand-Dependent Antagonism

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Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture

The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis.In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.The two defining characteristics of human embryonic stem cells (hESCs),¹ self-renewal and pluripotency, are maintained by a delicate balance of intracellular and extracellular signaling processes. Extracellular regulation is primarily the result of changes in the microenvironment surrounding the cells during growth in vitro or in vivo. HESCs interact with this “niche ” through support cells, extracellular matrix (ECM) components, and autocrine/paracrine signaling (reviewed in Refs. 1–3). Modulation of any of these supportive elements individually or in combination has been used extensively to alter hESC behavior (1–3).The culture of hESCs, as well as that of human induced pluripotent stem cells (hiPSCs), is conventionally performed on a layer of irradiated mouse embryonic fibroblast cells (MEFs). These MEFs are believed to promote the maintenance of hESCs and hiPSCs through the secretion of beneficial support proteins and cytokines into the soluble microenvironment. A number of proteomic studies have been conducted that examine the secretome of feeder-cell layers in an attempt to elucidate proteins and pathways essential for hESC and hiPSC survival (4–7). Alternatively, hESCs and hiPSCs can be cultured in feeder-free conditions in the absence of support cells. In feeder-free conditions, hESCs and hiPSCs are most often grown on the basement membrane matrix Matrigel™ in medium that has been previously conditioned by MEFs (MEF-CM). Matrigel™ is a gelatinous mixture that is secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (8). Although recent studies have proposed that a variety of defined matrices can support the growth of hESCs and hiPSCs, few of these can maintain a wide range of stem cell lines and therefore are typically not used in place of Matrigel™. The properties of Matrigel™ that make it such an effective matrix for hESC and hiPSC culture remain poorly understood. Because of the complexity of matrices like Matrigel™, the majority of proteomic studies that examine the hESC and hiPSC microenvironment have focused on contributions from support cells and soluble extracellular factors.The ECM is typically a complex network of structural proteins and glycosaminoglycans that function to support cells through the regulation of processes such as adhesion and growth factor signaling (9). Thus, it is not surprising that the generation of a well-defined matrix capable of facilitating hESC and hiPSC self-renewal has remained difficult (10). Previous proteomic investigations of Matrigel™ and other matrices supportive of hESC maintenance in vitro have revealed the presence of numerous growth, binding, and signaling proteins (11, 12). Further examination of how hESCs and hiPSCs interact with these complex matrices would provide critical information about what role the ECM plays in the organization of processes involved in the regulation of self-renewal and pluripotency.A recent study has established the ability of hESC-derived matrix microenvironments to alter tumorigenic properties through the reprogramming of metastatic melanoma cells (13). Importantly, this effect was found to be dependent on the exposure of metastatic cells to hESC-derived conditioned Matrigel™. Culture of metastatic melanoma cells in hESC-conditioned medium did not promote the reprogramming effect. These data suggest that the proteins responsible for this effect were integrated in the matrix. With the use of immunochemical techniques, it was later found that the left-right determination (Lefty) proteins A and B that were deposited in the matrix by hESCs during conditioning were at least in part responsible for the cellular change observed in metastatic cells (14). The Lefty A and B proteins are antagonists of transforming growth factor (TGF)-β signaling that act directly on Nodal protein, a critical regulator of the stem cell phenotype (15, 16). Subsequent studies of conditioned matrix utilizing mESCs implicated the bone morphogenic protein (BMP) 4 antagonist Gremlin as a primary regulator of the observed changes in metastatic cells (17). Collectively, these studies were all biased by a targeted analysis of potential effectors of metastatic cells. A comprehensive proteomic analysis of conditioned matrix could potentially reveal other factors involved in metastatic cell reprogramming. Furthermore, proteomic examination of hESC and hiPSC conditioned matrix could expose factors important in the regulation of self-renewal and pluripotency by the microenvironment in vitro.To this end, we have analyzed both types of human pluripotent stem cells, hESCs and hiPSCs, via a mass spectrometry (MS)-based proteomics approach to identify proteins deposited during growth in feeder-free conditions in vitro on Matrigel™. To investigate the hESC- and hiPSC-derived matrix, the metabolic labeling technique known as stable isotope labeling with amino acids in cell culture (SILAC) was used (18). SILAC facilitates the identification of hESC- and hiPSC-derived proteins that would otherwise be confounded by the presence of mouse-derived protein background from Matrigel™. From the proteomic analysis of three cells lines, namely, the hESC lines H9 and CA1 and the hiPSC line BJ-1D, we identified a total of 621, 1355, and 1350 total unique proteins, respectively. This work represents the first analysis of a hESC- and hiPSC-derived conditioned matrix and resulted in the identification of at least one novel microenvironmental contributor responsible for the regulation of human pluripotent stem cells.     

Mass Spectrometry Based Glycoproteomics—From a Proteomics Perspective

Glycosylation is one of the most important and common forms of protein post-translational modification that is involved in many physiological functions and biological pathways. Altered glycosylation has been associated with a variety of diseases, including cancer, inflammatory and degenerative diseases. Glycoproteins are becoming important targets for the development of biomarkers for disease diagnosis, prognosis, and therapeutic response to drugs. The emerging technology of glycoproteomics, which focuses on glycoproteome analysis, is increasingly becoming an important tool for biomarker discovery. An in-depth, comprehensive identification of aberrant glycoproteins, and further, quantitative detection of specific glycosylation abnormalities in a complex environment require a concerted approach drawing from a variety of techniques. This report provides an overview of the recent advances in mass spectrometry based glycoproteomic methods and technology, in the context of biomarker discovery and clinical application.With recent advances in proteomics, analytical and computational technologies, glycoproteomics—the global analysis of glycoproteins—is rapidly emerging as a subfield of proteomics with high biological and clinical relevance. Glycoproteomics integrates glycoprotein enrichment and proteomics technologies to support the systematic identification and quantification of glycoproteins in a complex sample. The recent development of these techniques has stimulated great interest in applying the technology in clinical translational studies, in particular, protein biomarker research.While glycomics is the study of glycome (repertoire of glycans), glycoproteomics focuses on studying the profile of glycosylated proteins, i.e. the glycoproteome, in a biological system. Considerable work has been done to characterize the sequences and primary structure of the glycan moieties attached to proteins (1–3), and their structural alterations related to cancer (4–6). Recent reports have provided a comprehensive overview of the concept of glycomics and its prospective in biomarker research (7–10). In contrast, this review is focused on recent developments in glycoproteomic techniques and their unique application and technical challenge to biomarker discovery.

Glycoproteomics in Biomarker Discovery and Clinical Study

Most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked glycans with diverse structures (2). The glycosylation form of a glycoprotein is highly specific at each glycosylation site and generally stable for a given cell type and physiological state. However, the glycosylation form of a protein can be altered significantly because of changes in cellular pathways and processes resulting from diseases, such as cancer, inflammation, and neurodegeneration. Such disease-associated alterations in glycoproteins can happen in one or both of two ways: 1) protein glycosylation sites are either hypo, hyper, or newly glycosylated and/or; 2) the glycosylation form of the attached carbohydrate moiety is altered. In fact, altered glycosylation patterns have long been recognized as hallmarks in cancer progression, in which tumor-specific glycoproteins are actively involved in neoplastic progression and metastasis (5, 6, 11, 12). Sensitive detection of such disease-associated glycosylation changes and abnormalities can provide a unique avenue to develop glycoprotein biomarkers for diagnosis and prognosis. In addition, intervention in the glycosylation and carbohydrate-dependent cellular pathways represent a potential new modality for cancer therapies (6, 11, 13). 14, 15) that are glycosylated proteins or protein complexes.

Table I

Listing of some of the US Food and Drug Administration (FDA) approved cancer biomarkers

MARMoSET – Extracting Publication-ready Mass Spectrometry Metadata from RAW Files

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Highlights

• •Automated metadata extraction from potentially large sets of mass spectrometric raw data.
• •Reduction of extracted metadata into groups of shared parameter sets.
• •Tabular representation for quality control, reporting and publication.

     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

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.     

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (���¦æ�)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhK''s activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.In the cascade activation of glycogenolysis in skeletal muscle, phosphorylase kinase (PhK),¹ upon becoming activated through phosphorylation, subsequently phosphorylates glycogen phosphorylase in a Ca²⁺-dependent reaction. This phosphorylation of glycogen phosphorylase activates its phosphorolysis of glycogen, leading to energy production (1). The 1.3 MDa (αβγδ)₄ PhK complex was the first protein kinase to be characterized and is among the largest and most complex enzymes known (2). As such, the intact complex has proved to be refractory to high resolution x-ray crystallographic or NMR techniques; however, low resolution structures of the nonactivated and Ca²⁺-saturated conformers of PhK have been deduced through modeling (3) and solved by means of three-dimensional electron microscopic (EM) reconstruction (4–7), and they show that the complex is a bilobal structure with interconnecting bridges. Approximate locations of small regions of each subunit in the complex are known (8–10) and show that the subunits pack head-to-head as apparent αβγδ protomers that form two octameric (αβγδ)₂ lobes associating in D₂ symmetry (11), although direct evidence that the αβγδ protomers are discrete, functional subcomplexes has been lacking until now.Approximately 90% of the mass of the PhK complex is involved in its regulation. Its kinase activity is carried out by the catalytic core of the γ subunit (44.7 kDa), with the k_cat being enhanced up to 100-fold by multiple metabolic, hormonal, and neural stimuli that are integrated through allosteric sites on PhK''s three regulatory subunits, α, β, and δ (12). The small δ subunit (16.7 kDa), which is tightly bound integral calmodulin (13), binds to at least the C-terminal regulatory domain of the γ subunit (γCRD) (14, 15), thereby mediating activation of the catalytic subunit by the obligate activator Ca²⁺ (16). The α and β subunits, as deduced from DNA sequencing, are polypeptides of 1237 and 1092 amino acids, respectively, with calculated masses prior to post-translational modifications of 138.4 and 125.2 kDa (17, 18). Both subunits can be phosphorylated by numerous protein kinases, including cAMP-dependent protein kinase and PhK itself (2). The α and β subunits are also homologous (38% identity and 61% similarity); however, each subunit has unique phosphorylatable regions that contain nearly all the phosphorylation sites found in these subunits (17, 18).The regulation of PhK activity by both Ca²⁺ (19–23) and phosphorylation has been studied extensively (reviewed in Ref. 24); however, only the structural effects induced by Ca²⁺ are well characterized (25), primarily through comparison of the non-activated and Ca²⁺-activated conformers using three-dimensional EM reconstructions (4), small angle x-ray scattering modeling (3), and biophysical (26–28) and chemical crosslinking methods (29–32). In contrast to the Ca²⁺-activated versus non-activated conformers, there are no reported structures of phosphorylated PhK to compare against the non-activated form. A very small amount of structural information for phospho-activated PhK derived from chemical crosslinking raises the possibility of phosphorylation-dependent communication between the β and γ subunits: Arg-18 in the N-terminal phosphorylatable region of β was found to be relatively near the γCRD (33). Several lines of evidence suggest that transduction of the activating phosphorylation signal in PhK occurs concomitantly with conformational changes in β (33) that are detected via various methods (10, 34), including chemical crosslinking (35). For example, crosslinking of only the phosphorylated conformer by the short-span crosslinker 1,5-difluoro-2,4-dinitrobenzene results in the formation of β homodimers (35). Correspondingly, more recent two-hybrid screens of the full length β subunit against itself yielded positive binding interactions only for point mutants in which the N-terminal phosphorylatable serine residues were mutated to phosphomimetic glutamates (33). It should be noted, however, that both chemical crosslinking and two-hybrid screening have potential drawbacks in the study of subunit interactions within a multisubunit complex. In the case of the latter, it is difficult when observing homodimeric two-hybrid interactions to determine whether they correspond to naturally occurring interactions between two like subunits within a complex or between two interacting regions within a single subunit of that complex. Studying subunit interactions in a complex through chemical crosslinking comes with its own inherent limitations. For example, an initial mono-derivatization can potentially cause a conformational change in one subunit that might affect the subsequent crosslinking reaction. This is particularly the case if the crosslinker contains a functionality, such as an aromatic group, that can unexpectedly direct it to a specific locus on the protein complex (36, 37). In addition, the spacer arms on many crosslinkers are sufficiently long to confound interpretation as to whether two subunits within a complex are actually in contact. Similarly, it should be proved that any observed crosslinked conjugate is formed from subunits within a complex, as opposed to between complexes (38, 39), a control that is often not run. Thus, it is prudent to analyze subunit interactions within a complex using a variety of approaches.To corroborate, complement, and expand the previous two-hybrid screening and chemical crosslinking studies of PhK''s subunit interactions and to investigate changes in the pattern of subunit interactions induced by phosphorylation, we carried out comparative MS analyses of both intact and partially denatured forms of nonactivated and phospho-activated PhK using mass spectrometers modified specifically to enhance the transmission of large noncovalently bound protein complexes (40–42). The array of subunit interactions detected for the nonactivated PhK complex largely replicated those reported in the crosslinking literature for this conformer, both corroborating those earlier studies and validating the use of these MS approaches to study subunit interactions within the PhK complex. Additionally, several novel subcomplexes of PhK were revealed, most notably an αβγδ protomer, which corroborates the observed packing of this subcomplex in the D₂ symmetrical (αβγδ)₄ native complex (9, 11). Moreover, we show herein that the array of subunit interactions detected for phospho-activated PhK differs significantly from that observed for the nonactivated conformer, with only the former showing extensive self-interactions between and among the regulatory β subunits. As is discussed, this suggests that activation through phosphorylation is associated with increased interprotomeric interactions in the bridged core of the PhK complex (33, 35).     

Detection of Molecular Ions of Cerebroside by Gas Chromatography—Mass Spectrometry

Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX-53. The molecular weight of the enzyme was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH 4.7 by isoelectric focusing. This enzyme contained 1.12 atoms of manganese and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzyme was 9.5 mM for oxalic acid at the optimal pH 4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I^–, Br^–, semicarbazide, and hydroxylamine.     

The mzQuantML Data Standard for Mass Spectrometry–based Quantitative Studies in Proteomics

The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS)¹ leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases. In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative http://www.psidev.info/mzquantml.The Proteomics Standards Initiative (PSI) has been working for ten years to improve the reporting and standardization of proteomics data. The PSI has published minimum reporting guidelines, called MIAPE (Minimum Information about a Proteomics Experiment) documents, for MS-based proteomics (1) and molecular interactions (2), as well as data standards for raw/processed MS data in mzML (3), peptide and protein identifications in mzIdentML (4), transitions for selected reaction monitoring analysis in TraML (5), and molecular interactions in PSI-MI format (6). Standards are particularly important for quantitative proteomics research, because the associated bioinformatics analysis is highly challenging as a result of the range of different experimental techniques for deriving abundance values for proteins using MS. The techniques can be broadly divided into those based on (i) differential labeling, in which a metabolic label or chemical tag is applied to cells, peptides, or proteins, samples are mixed, and intensity signals for peptide ions are compared within single MS runs; or (ii) label-free methods in which MS runs occur in parallel and bioinformatics methods are used to extract intensity signals, ensuring that like-for-like signals are compared between runs (7). In most label-based and label-free approaches, peptide ratios or abundance values must be summarized in order for one to arrive at relative protein abundance values, taking into account ambiguity in peptide-to-protein inference. Absolute protein abundance values can typically be derived only using internal standards spiked into samples of known abundance (8, 9). The PSI has recently developed a MIAPE-Quant document defining and describing the minimal information necessary in order to judge or repeat a quantitative proteomics experiment.Software packages tend to report peptide or protein abundance values in a bespoke format, often as tab or comma separated values, for import into spreadsheet software. In complementary work, the PSI has developed a standard format for capturing these final results in a standardized tab separated value format, called mzTab, suitable for post-processing and visualization in end-user tools such as Microsoft Excel or the R programming language. The final results of a quantitative analysis are sufficient for many purposes, such as performing statistical analysis to determine differential expression or cluster analysis to find co-expressed proteins. However, mzTab (or similar bespoke formats) was not designed to hold a trace of how the peptide and protein abundance values were calculated from MS data (i.e. metadata is lost that might be crucial for other tasks). For example, most quantitative software packages detect and quantify so-called “features” (representing all ions collected for a given peptide) in two-dimensional MS data, where the two dimensions are retention time from liquid chromatography (LC) and mass over charge (m/z). Without capturing the two-dimensional coordinates of the features, it is not possible to write visualization software showing exactly what the software has quantified; researchers have to trust that the software has accurately quantified all ions from isotopes of a given peptide, excluding any overlapping ions derived from other peptides. The history of proteomics research has been one in which studies of highly variable quality have been published. There is also little quality control or benchmarking performed on quantitative software (10), meaning it is difficult to make quality judgments on a set of peptide and protein abundance values. The PSI has recently developed mzML, which can capture raw or processed MS data in a vendor neutral format, and the mzIdentML standard, to capture search engine results and the important metadata (such as software parameters), such that peptide and protein identification data can be interpreted consistently. These two standards are now being used for data sharing and to support open source software development, so that informatics groups can focus on algorithmic development rather than file format conversions. Until now, there has been no widely used open source format or data standard for capturing metadata and data relating to the quantitation step of analysis pipelines. In this work, we report the mzQuantML standard from the PSI, which has recently completed the PSI standardization process (11), from which version 1.0 was released. We believe that quantitative proteomics research will benefit from improved capabilities for tracing what manipulations have happened to data at each stage of the analysis process. The mzQuantML standard has been designed to store quantitative values calculated for features, peptides, proteins, and/or protein groups (where there is ambiguity in protein inference), plus associated software parameters. It has also been designed to accommodate small molecule data to improve interoperability with metabolomics investigations. The format can represent experimental replicates and grouping of replicates, and it has been designed via an open and transparent process.