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Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-[beta] superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-[beta] signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF-[beta] signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome., Introduction Bronchopulmonary dysplasia (BPD) is a lung disease that disproportionately affects infants born at less than 28 weeks gestation, the threshold for extreme prematurity (1). Despite multiple medical advances including [...]

Author(s): Zhixun Dou [1]; Kanad Ghosh [1]; Maria Grazia Vizioli [2, 3]; Jiajun Zhu [1]; Payel Sen [1]; Kirk J. Wangensteen [4]; Johayra Simithy [1, 5]; Yemin Lan [1]; Yanping [...]

Author(s): Gloria Jih [1, 2]; Nahid Iglesias [1, 2]; Mark A. Currie [1, 2]; Natarajan V. Bhanu [3]; Joao A. Paulo [1]; Steven P. Gygi [1]; Benjamin A. Garcia [3]; [...]

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses (1). Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses (2). Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones (3). Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles (4,5), it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses (6,7). We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signalling., As viruses commandeer cellular functions to promote viral production, they induce numerous cellular changes. Manipulation of host chromatin is important for viral takeover of cellular functions (1,8-11). Although there are [...]

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs) (1,2), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2-4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. [Cbfa2t2.sup.-/-] mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency., The germ line first segregates from somatic lineages via the specification of PGCs between embryonic day (E)6.25-7.25 in mice (1,2). PRDM14 regulates pluripotency (5-7), and is the only known transcription [...]

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways., A majority of B cell lymphomas-including follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), the most common forms of non-Hodgkin lymphoma-arise from germinal center (GC) B cells. GCs [...]

ABSTRACT The aim of this study was to test the effect of copper supplementation in a formulated feed on the growth, feed efficiency, and nutritional composition of subadults Octopus vulgaris, [...]

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics (1). The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours (2-4). However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Rasdriven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling., The Ras family of oncogenes is activated in a large fraction of human cancers (5). To explore possible connections between KMTs and human cancers driven by activation of Ras, we [...]

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing (1,2). SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. [...]

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating [CD45-C-KIT .sup.low/+]TIE2+ bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process., Exosomes are small membrane vesicles (30-100 nm) derived from the luminal membranes of multivesicular bodies and constitutively released by fusion with the cell membrane (1-5). In addition to diffusible factors, [...]

A central hub of carbon metabolism is the tricarboxylic acid cycle (1), which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which tricarboxylic acid metabolism plays a minor role (2). Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen (3), yet the parasite genome encodes all of the enzymes necessary for a complete tricarboxylic acid cycle (4). Here, by tracing [sup.13]C-labelled compounds using mass spectrometry (5), we show that tricarboxylic acid metabolism in the human malaria parasite Plasmodium falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture from the canonical textbook pathway. We find that this pathway is not cyclic, but rather is a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction, thereby generating two-carbon units in the form of acetyl-coenzyme A. We further show that glutamine-derived acetyl-coenzyme A is used for histone acetylation, whereas glucose-derived acetyl-coenzyme A is used to acetylate amino sugars. Thus, the parasite has evolved two independent production mechanisms for acetyl-coenzyme A with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments., The mitochondrion of P. falciparum contains the smallest genome sequenced to date, and seems to have evolved reduced functional roles compared with other eukaryotic organisms (6). Moreover, the limited number [...]

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability (1). Causal mutations have been found in approximately 90 X-linked genes (2); however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of ~7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways (3). Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR., XLMR affects 1-4 out of 2,000 males, causing intellectual disability (intelligence quotient (IQ) PHF8 belongs to a subfamily of JmjC domain-containing proteins that also includes KIAA1718 (also known as JHDM1D) [...]

The goal of this research project was to investigate the long-term effects of whole-body vibration (WBV) training on force production. Thirty-one female basketball players were randomly distributed in an experimental group: VG (vibration) and a control group: CG (no vibration). Both groups participated in the same training program; however, the experimental group (VG) performed a set of exercises on a vibration platform (Power Plate[R]) at 30- to 35-Hz frequency and 4 mm amplitude, whereas the CG performed the same exercises at 0 Hz. Muscle performance of the legs was tested on a contact-time platform (Ergojump[R], Finland) through several tests: squat jump (S J), countermovement jump (CMJ), and 15-second maximal performance jump; squat leg power (knee extension) was also evaluated using an Ergopower machine[R] (Bosco, Italy). After 14 weeks, there was a significant increase (p < 0.001) from initial to final tests in both groups (CG and VG) in S J, CMJ, 15 seconds, and squat leg power. However, there were no significant differences between the VG and the CG for any of the parameters evaluated. The findings of this study indicate that WBV training has no additive or discernible effect on the strength development of female basketball players after several weeks of use, suggesting that the application of this technology has no advantages over traditional strength training methods. Key Words physical training, strength, women

J Strength Cond Res 24(1): 165-1 70, 2010--To examine the relationship between metabolic (i.e., blood lactate concentration) and perceptual (ratings of perceived exertion, RPE) responses and their association with variables describing the characteristics of the singles tennis match play. Eight trained and internationally ranked (Association of Tennis Professionals rankings) male tennis players were studied during singles matches (best of 3 sets) played on an outdoor clay court surface during a professional invitational tournament. Ratings of perceived exertion and blood lactate concentrations were determined at selected changeovers during the game. The variables describing the characteristics of the matches, (a) duration of rallies (DR), (b) rest time, (c) effective playing time, and (d) strokes per rally (SR), were determined from video recordings. Significant correlations (r = 0.57-0.48; p < 0.01) were found for RPE-blood lactate responses during the games. Both RPE and blood lactate concentration values were significantly correlated with SR and DR (r = 0.80-0.61; p < 0.001) in service games. Average RPE was significantly higher (p < 0.05) following service games (13.5 [+ or -] 1.9; n = 24) than following receiving games (12.2 [+ or -] 2.0; n = 22). Mean blood lactate concentrations were significantly higher (p < 0.05) following service games (4.4 [+ or -] 2.4 mmol x [L.sup.-1]; n = 24) than following receiving games (3.0 [+ or -] 1.3 mmol x [L.sup.-1]; n = 22). These results indicate that monitoring RPE may be a useful technique for regulating on-court tennis training intensity. Moreover, blood lactate may mediate the relation between RPE and tennis match play intensity. KEY WORDS tennis competition play, blood lactate concentration, rating of perceived exertion

Covalent adenosine diphosphate (ADP)-ribosylation of proteins is a post-translational modification that can occur both enzymatically and nonenzymatically and has been linked to many biological processes, such as the DNA damage and response mechanisms. Although the biochemistry of protein ADP-ribosylation has been extensively studied, the identification of physiological substrates remains a significant challenge due to inadequate tools for characterizing these modified peptides. Here we show that the use of electron transfer dissociation (ETD) greatly simplifies the mass spectrometric (MS) sequencing of ADP-ribosylated peptides over the conventional collisionally activated dissociation (CAD) approach. ETD sequencing of ADP-ribosylated peptides can be performed within a time scale compatible with nanoflow liquid chromatography and should prove useful for high-throughput large-scale studies on this unique class of modified peptides. 10.1021/ac902134y

Quantitative studies of protein abundance rarely span more than a small number of experimental conditions and replicates. In contrast, quantitative studies of transcript abundance often span hundreds of experimental conditions and replicates. This situation exists, in part, because extracting quantitative data from large proteomics datasets is significantly more difficult than reading quantitative data from a gene expression microarray. To address this problem, we introduce two algorithmic advances in the processing of quantitative proteomics data. First, we use space-partitioning data structures to handle the large size of these datasets. Second, we introduce techniques that combine graph-theoretic algorithms with space-partitioning data structures to collect relative protein abundance data across hundreds of experimental conditions and replicates. We validate these algorithmic techniques by analyzing several datasets and computing both internal and external measures of quantification accuracy. We demonstrate the scalability of these techniques by applying them to a large dataset that comprises a total of 472 experimental conditions and replicates. kd-tree | orthogonal range query | quantitative proteomics | space partitioning data structures | tandem mass spectrometry

Acetylation is a well-studied posttranslational modification that has been associated with a broad spectrum of biological processes, notably gene regulation. Many studies have contributed to our knowledge of the enzymology underlying acetylation, including efforts to understand the molecular mechanism of substrate recognition by several acetyltransferases, but traditional experiments to determine intrinsic features of substrate site specificity have proven challenging. Here, we combine experimental methods with clustering analysis of protein sequences to predict protein acetylation based on the sequence characteristics of acetylated lysines within histones with our unique prediction tool PredMod. We define a local amino acid sequence composition that represents potential acetylation sites by implementing a clustering analysis of histone and nonhistone sequences. We show that this sequence composition has predictive power on 2 independent experimental datasets of acetylation marks. Finally, we detect acetylation for selected putative substrates using mass spectrometry, and report several nonhistone acetylated substrates in budding yeast. Our approach, combined with more traditional experimental methods, may be useful for identifying acetylated substrates proteome-wide. histone | nonhistone | prediction | acetylation | PredMod

A combination of immobilized metal affinity chromatography and tandem mass spectrometry is used to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. The methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.

Histones are the fundamental components of the nucleosome. Physiologically relevant variation is introduced into this structure through chromatin remodeling, addition of covalent modifications, or replacement with specialized histone variants. The histone H3 family contains an evolutionary conserved variant, H3.3, which differs in sequence in only five amino acids from the canonical H3, H3.1, and was shown to play a role in the transcriptional activation of genes. Histone H3.3 contains a serine (S) to alanine (A) replacement at amino acid position 31 (S31). Here, we demonstrate by both MS and biochemical methods that this serine is phosphorylated (S31P) during mitosis in mammalian cells. In contrast to H3 S10 and H3 S28, which first become phosphorylated in prophase, H3.3 S31 phosphorylation is observed only in late prometaphase and metaphase and is absent in anaphase. Additionally, H3.3 S31P forms a speckled staining pattern on the metaphase plate, whereas H3 S10 and H3 S28 phosphorylation localizes to the outer regions of condensed DNA. Furthermore, in contrast to phosphorylated general H3, H3.3 S31P is localized in distinct chromosomal regions immediately adjacent to centromeres. These findings argue for a unique function for the phosphorylated isoform of H3.3 that is distinct from its suspected role in gene activation. mitosis | cell cycle | subtype modification

A new method of sample preparation was developed for MALDI-TOF-MS analysis of oligonucleotides. First, aqueous DNA samples are dispensed and allowed to dry. Then 6-aza-2-thiothymine matrix dissolved in nonaqueous volatile solvents is applied on top of the DNA residue to form a thin homogeneous film. MALDI-TOF analysis shows such preparation generates much better shot-to-shot and sample-to-sample reproducibility and essentially eliminates the need to search for 'hot' spots. The increased homogeneity of the matrix/analyte crystal distribution results in significant improvement for quantitative and high-throughput analyses of DNA. Using this method, isotopically resolved oligonucleotide spectra up to a 24-mer can also be easily obtained in a reflectron instrument. Due to the ease of preparation, this method could be widely useful for a number of applications such as for assays that are performed on surface in vitro, as the thin-layer matrix could be applied directly for MALDI analysis.

The apparent mass resolution of oligonucleotides in time-of-flight (TOF) mass spectrometers has been examined. In a reflectron TOF instrument, where the isotopic profile can be completely resolved, the apparent resolution matches the instrument's resolving power. In a linear TOF instrument, unresolved isotopic profiles limit the apparent resolution to much lower values than the actual instrument resolution. By using [sup.12]C/[sup.14]N-enriched oligonucleotides, the apparent resolution can be improved significantly. The isotope enrichment method also enhances the signal-to-noise ratio.

Ever since Stephen Pagets 1889 hypothesis, metastatic organotropism has remained one of cancers greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins [sub.6][sub.4] and [sub.6][sub.1] were associated with lung metastasis, while exosomal integrin [sub.v][sub.5] was linked to liver metastasis. Targeting the integrins [sub.6][sub.4] and [sub.v][sub.5] decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis., Author(s): Ayuko Hoshino [1]; Bruno Costa-Silva [1]; Tang-Long Shen [1, 2]; Goncalo Rodrigues [1, 3]; Ayako Hashimoto [1, 4]; Milica Tesic Mark [5]; Henrik Molina [5]; Shinji Kohsaka [6]; Angela [...]

Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis- suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin-dependent epithelial traits and Sec23a-mediated tumor cell secretome., Early events of metastatic dissemination are thought to be initiated by epithelial-to-mesenchymal transition (EMT) in carcinoma cells, promoting tumor cell migration and invasion (1-3). Although the involvement of EMT in [...]

Author(s): Héctor Peinado; Ma[s caron]a Alec[caron]kovi[c acute]; Simon Lavotshkin; Irina Matei; Bruno Costa-Silva; Gema Moreno-Bueno; Marta Hergueta-Redondo; Caitlin Williams; Guillermo García-Santos; Cyrus M Ghajar; Ayuko Nitadori-Hoshino; Caitlin Hoffman; Karen Badal; [...]

A report that HDAC3 is a critical, transcription-independent regulator of mitosis is presented. Phosphorylation of H2S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin.

Author(s): Wolfgang Fischle (corresponding author) [1]; Boo Shan Tseng [2]; Holger L. Dormann [1]; Beatrix M. Ueberheide [3]; Benjamin A. Garcia [3]; Jeffrey Shabanowitz [3]; Donald F. Hunt [3, 4]; [...]