Human Protein Database Research Articles

Sensitive, Simple, and Robust Nano-Liquid Chromatography-Mass Spectrometry Method for Amyloid Protein Subtyping.

Amyloidosis is a rare condition characterized by deposits of insoluble proteins in the form of β-pleated sheets. These deposits interfere with the normal structure and function of varying tissues. Thirty-one amyloid proteins have been identified, and the correct identification is critical due to the varying treatments. Immunohistochemistry, the most routine method for identification of amyloid proteins, suffers from limitations. Mass spectrometry (MS)-based methods offer better sensitivity and specificity. We describe here a sensitive, simple, and robust MS-based method for the identification of amyloid proteins. Amyloid deposits are excised from formalin-fixed tissue by laser microdissection and is put through protein extraction followed by trypsin digestion. The resulting peptides are separated by nano-liquid chromatography and analyzed by high-resolution Orbitrap mass spectrometry. The mass spectrometry data are then searched against a human protein database for identification and semi-quantification.

SCMMTP: identifying and characterizing membrane transport proteins using propensity scores of dipeptides.

BackgroundIdentifying putative membrane transport proteins (MTPs) and understanding the transport mechanisms involved remain important challenges for the advancement of structural and functional genomics. However, the transporter characters are mainly acquired from MTP crystal structures which are hard to crystalize. Therefore, it is desirable to develop bioinformatics tools for the effective large-scale analysis of available sequences to identify novel transporters and characterize such transporters.ResultsThis work proposes a novel method (SCMMTP) based on the scoring card method (SCM) using dipeptide composition to identify and characterize MTPs from an existing dataset containing 900 MTPs and 660 non-MTPs which are separated into a training dataset consisting 1,380 proteins and an independent dataset consisting 180 proteins. The SCMMTP produced estimating propensity scores for amino acids and dipeptides as MTPs. The SCMMTP training and test accuracy levels respectively reached 83.81% and 76.11%. The test accuracy of support vector machine (SVM) using a complicated classification method with a low possibility for biological interpretation and position-specific substitution matrix (PSSM) as a protein feature is 80.56%, thus SCMMTP is comparable to SVM-PSSM. To identify MTPs, SCMMTP is applied to three datasets including: 1) human transmembrane proteins, 2) a photosynthetic protein dataset, and 3) a human protein database. MTPs showing α-helix rich structure is agreed with previous studies. The MTPs used residues with low hydration energy. It is hypothesized that, after filtering substrates, the hydrated water molecules need to be released from the pore regions.ConclusionsSCMMTP yields estimating propensity scores for amino acids and dipeptides as MTPs, which can be used to identify novel MTPs and characterize transport mechanisms for use in further experiments.Availabilityhttp://iclab.life.nctu.edu.tw/iclab_webtools/SCMMTP/

Genome wide association and genomic prediction for growth traits in juvenile farmed Atlantic salmon using a high density SNP array.

BackgroundThe genetic architecture of complex traits in farmed animal populations is of interest from a scientific and practical perspective. The use of genetic markers to predict the genetic merit (breeding values) of individuals is commonplace in modern farm animal breeding schemes. Recently, high density SNP arrays have become available for Atlantic salmon, which facilitates genomic prediction and association studies using genome-wide markers and economically important traits. The aims of this study were (i) to use a high density SNP array to investigate the genetic architecture of weight and length in juvenile Atlantic salmon; (ii) to assess the utility of genomic prediction for these traits, including testing different marker densities; (iii) to identify potential candidate genes underpinning variation in early growth.ResultsA pedigreed population of farmed Atlantic salmon (n = 622) were measured for weight and length traits at one year of age, and genotyped for 111,908 segregating SNP markers using a high density SNP array. The heritability of both traits was estimated using pedigree and genomic relationship matrices, and was comparable at around 0.5 and 0.6 respectively. The results of the GWA analysis pointed to a polygenic genetic architecture, with no SNPs surpassing the genome-wide significance threshold, and one SNP associated with length at the chromosome-wide level. SNPs surpassing an arbitrary threshold of significance (P < 0.005, ~ top 0.5 % of markers) were aligned to an Atlantic salmon reference transcriptome, identifying 109 SNPs in transcribed regions that were annotated by alignment to human, mouse and zebrafish protein databases. Prediction of breeding values was more accurate when applying genomic (GBLUP) than pedigree (PBLUP) relationship matrices (accuracy ~ 0.7 and 0.58 respectively) and 5,000 SNPs were sufficient for obtaining this accuracy increase over PBLUP in this specific population.ConclusionsThe high density SNP array can effectively capture the additive genetic variation in complex traits. However, the traits of weight and length both appear to be very polygenic with only one SNP surpassing the chromosome-wide threshold. Genomic prediction using the array is effective, leading to an improvement in accuracy compared to pedigree methods, and this improvement can be achieved with only a small subset of the markers in this population. The results have practical relevance for genomic selection in salmon and may also provide insight into variation in the identified genes underpinning body growth and development in salmonid species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2117-9) contains supplementary material, which is available to authorized users.

Abstract 1088: Exploration of the human peptidome for cancer-specific peptide-MHC targets

Abstract Introduction MHC class-I molecules display peptide fragments of intracellular proteins on the cell surface. Peptide-MHC (pMHC) complexes containing peptide fragments derived from antigenic proteins lead to a T-cell mediated immune response. We are interested in cancer-specific pMHC complexes that are targetable by therapeutic molecules, e.g., bispecific antibodies. A significant concern is off-target toxicity of the therapeutic molecules due to pMHC complexes similar to the target complex in essential, normal tissues. Thus we performed a comprehensive exploration of the 9-mer human intracellular peptidome to computationally identify potential breast cancer-specific pMHC class I targets and evaluate the likelihood of cross-reactivity. Methods Our method has four steps: 1. We first identified amino acid sequences of all intracellular proteins and 9-mers from a human protein database (UniprotKB). 2. Next, we use a computational program to predict binding affinities and half-lives of all possible combinations of 9-mers and 13 HLA-A and HLA-B alleles encompassing major HLA supertypes; combinations passing affinity and half-life criteria are classified as potential complexes. 3. For each potential pMHC complex, we identify potential similar complexes; two complexes are considered similar if they have the same HLA allele and 5 or more identical amino acids at identical positions. 4. We classify a complex as a potential target if combined mRNA expression of the complex, and similar complexes is higher in breast cancer tissues (TCGA data) compared to normal essential tissues (GTEx data) in at least 5% of the cancer samples. Results We extracted all intracellular protein sequences available in the UniprotKB, from which all 9-mer peptides were derived. Hundreds of thousands of potential pMHC complexes using binding predictions for 13 HLA A and B alleles were identified. For each potential MHC binding peptide, similar amino acid sequences were identified. Combined mRNA expression analysis resulted in the discovery of potential targets, which we further prioritized on the basis of likelihood of off-target effects and breast cancer expression levels of the peptide's gene(s) of origin. While we identified several thousand potential breast cancer specific targets, the number of targets with low likelihood of cross-reactivity is incredibly small. Conclusions We have developed a method to identify potential cancer-specific pMHC complexes and to predict potential off-targets of the drugs targeting them, which is essential to the development of safe drugs. Using this method, we examined the landscape of potential breast cancer specific targets derived from the human peptidome. Our examination showed that finding targets with low likelihood of cross-reactivity may be extremely difficult. Nonetheless, our method could help in prioritizing pMHC targets based on the evaluation of the potential cross-reactivity risk. Citation Format: Ankur Dhanik, Wen Zhang, Jessica Kirshner, Douglas MacDonald, Susan Croll, Calvin Lin, Gavin Thurston, Andrew Murphy. Exploration of the human peptidome for cancer-specific peptide-MHC targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1088. doi:10.1158/1538-7445.AM2015-1088

Evaluation of Cross-Sample Contamination in Tissue Microarrays by Polymerase Chain Reaction

In the past decade, the popularity and power of Tissue Microarray (TMA) technology has increased since it provides a method to detect diagnostic and prognostic markers in an array of clinical tissue specimens collected for translational research. TMAs allow for rapid and cost-effective analysis of hundreds of molecular markers at the nucleic acid and protein levels. This technology is particularly useful in the realization of the Human Protein Atlas Project, since it aims to create a reference database of non-redundant human proteins. In this context, it is important to assure the lack of cross-sample contamination due to the repeated use of the same needle in consecutive coring. Here we show that carry-over contamination from one tissue core to another does not occur, reinforcing the accuracy of the TMA technology in the simultaneous testing of multiple bio-samples.

Differences in serum protein 2D gel electrophoresis patterns of Przewalski's (Mongolian wild horse) and thoroughbred horses.

The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three proteins (a haptoglobin-2 alpha glycoprotein and two haptoglobin-2beta glycoproteins with different accession numbers) were downregulated in Przewalski's horse sera compared to thoroughbred horse sera. Moreover, two proteins (tetraspanin-18 and pM5) were upregulated in Przewalski's horses compared to thoroughbred horses. Haptoglobin-2 alpha and haptoglobin-2beta may serve as candidate molecules in future studies of inflammation, coagulation, immune modulation and pro-oxidant and antioxidant activity with consequential effects on the entire metabolism of the horse.

Abstract 5306: Shotgun proteomic analysis of human head and neck squamous cell carcinoma cell line SQ20B with diminished AHSG expression

Abstract The Alpha-Heremans-Schmid Glycoprotein (AHSG) has tumor promoting properties in animal models of breast cancer and lung cancer (1, 2). These cancer cells do not synthesize AHSG, instead utilizing the liver-generated glycoprotein that is abundant in serum. We have reported that head and neck squamous cell carcinoma (HNSCC) cell lines synthesize AHSG (in press) and have also detected abundant AHSG in primary HNSCC tumors (unpublished). Growth in serum-free medium and in vitro tumorigenic properties, including proliferation, adhesion and migration, are diminished in the HNSCC SQ20B cell line modified with AHSG-specific shRNA to express only twenty percent of the wild-type SQ20B cell line (SQ20B-AH20) compared to SQ20B modified with empty vector alone and expressing the wild-type amount of AHSG (SQ20B-EV) (in press). Here we have used shotgun proteomic analysis to identify additional proteins whose expression may also affect these in vitro properties of tumorigenesis associated with AHSG. The two cell lines were cultured in serum-free medium to avoid the contribution of exogenous AHSG in serum. Cell lysates were obtained, and the proteins were separated in polyacrylamide gels in duplicate. Each full protein lane was cut into 10 pieces, in-gel digested with trypsin and analyzed with tandem liquid chromatography mass spectrometry (LC-MS). MS data were analyzed against a recent human protein database. Proteins differentially expressed were quantified using a spectral counting approach. A total of 1386 distinct protein groups were identified in the two samples. Forty-eight proteins were differentially expressed in these samples (z score &amp;gt; 3, which corresponds to p ≤ 0.001, and Benjamini-Hochberg [statistical] FDR &amp;lt; 0.05). Proteins associated with the cytoskeleton, adhesion and apoptosis were over-represented in the group of differentially expressed proteins. Here we have shown that the human HNSCC cell line SQ20B exhibits changes in the proteome when AHSG expression is diminished. These proteins are critical for the tumorigenic properties of adhesion and migration. These data will help to identify mechanisms of tumorigenesis associated with AHSG in HNSCC.

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.

A128: Hierarchical Clustering Methodology for Exploration of Proteomic Profile in Tears: Seeking for Markers of Uveitis Associated With Juvenile Idiopathic Arthritis

Background/Purpose:Uveitis often is the only initial clinical manifestation of JIA for years. There are no reliable markers of JIA‐associated uveitis and impossibility to overcome ocular barriers impede diagnosis and treatment leading to blindness. During the last years the number of proteins identified in tears increased from 200 (Herber et al., 2001) of only 17 (Zhou et al., 2003) different molecular weights (MW) to 491 (de Souza et al., 2006) with 80 chemokines, cytokines, and growth factors (Sack et al., 2005). Objective of the study is to reveal protein markers of JIA‐associated uveitis in tears using high‐resolution mass‐spectrometry and hierarchical clustering methodology.Methods:Standard clinical protocol was used to diagnose uveitis and JIA. Tear samples drawn from 17 JIA‐patients with uveitis, 4 JIA‐patients without uveitis, 3 patients with systemic vasculitis, 4 patients with idiopathic uveitis and 3 healthy subjects were analyzed. Tears were collected on a filter paper, digested with trypsin and the peptides were purified on Zip tips. The samples were loaded to nano C18 column attached to Shimadzu nano LC coupled in‐line to LTQ Orbitrap XL tandem mass spectrometer (Thermo Fischer Scientific, GA, USA). The mass spectra of the peptides were detected with a data‐dependent 4‐event scan method (a survey FT‐MS parent scan followed by sequential datadependent FT‐MS/MS scans on the three most abundant peptide ions from the parent scan). Proteins were identified from the mass spectra results with Proteome Discoverer software for protein database search using the International Protein Index (IPI) Human Protein Database (version 1.79). Quantification was conducted using SIEVE.Results:About 3000 proteins were detected in tears. Among those, 236 proteins (MW 7.4–466 kDa) were chosen as candidates for early diagnosis of the JIA‐associated uveitis. Besides the rheumatoid factors RFET12, RF‐IP14, RF‐IP24, cytokines and T‐cell receptor alpha‐chain, we identified anti‐CD40 single‐chain antibody fragment A49, inhibiting protein CD40 which is a member of the TNF‐receptor superfamily, lipocalin‐1 precursor, associated with immune response and prostaglandin synthesis, clusterin inhibiting the cytolytic reaction of complement components, anti‐TNF‐alfa antibody light chain Fab fragment, protein phosphatase, regulatory subunit 15B, which plays a role in cell progression, apoptosis and regulation of membrane receptors and channels, DMBT/8kb.2 protein, which takes part in the interaction of tumor cells and the immune system.Conclusion:The result of our study demonstrates the benefits of high resolution mass‐ spectrometry for analysis and development of molecular signatures which can be used in diagnostics. Hierarchical clustering methodology allowed grouping according to expression profiles and the dendrogram construction procedure revealed clusters of proteins associated with iron metabolism as the most promising markers of JIA‐associated uveitis. Earlier it was shown that induced anterior uveitis in the eyes of rats can be described in terms of proteins drawn from the anterior chamber (Sobn et al., 2000), which result is close to our terms of description.

Diagnosis Of Amyloidosis Subtype By Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS) Proteomic Analysis

AimCorrect identification of the protein that is causing amyloidosis is crucial for clinical management. Current standard diagnostic methods have limited ability to detect the full range of amyloid forming proteins. We assessed combining specific sampling of amyloid deposits by LCM and analysis of tryptic digests by tandem MS proteomic analysis to determine whether the majority of amyloidosis clinical samples can be correctly classified as reported by the Mayo Clinic pathology department. MethodsWe studied 58 cases of well characterised amyloid deposition and 10 cases in which the amyloid subtype was unable to be diagnosed with confidence. For all specimens, 10µm sections of formalin-fixed paraffin embedded tissue were stained with Congo Red using a standard technique. LCM was performed using an Arcturus XT instrument with an infrared capture laser. Proteins were digested with trypsin and peptides were analysed by nano-liquid chromatography-coupled tandem mass spectrometry using a Chip CUBE-QTOF. Database searching was performed using Spectrum Mill (Agilent) with the NCBInr human protein database. Protein identification cut-offs were protein score > 11, peptide score > 10 and % scored peak intensity >60. ResultsBiopsy sites included: GIT (n=16), cardiac (n=12), soft tissue (n=8), renal (n=9), liver (n=3) and other (n=20). The amyloid subtype was able to be determined in 64 cases analysed. In 7 of these cases a second sample or second LCM was required as the first analysis was non-diagnostic. In 4 cases the amyloidogenic protein was not identified mostly due to the amyloid deposits being very small. Proteins identified included immunoglobulin light chain (localised amyloid n=6, systemic AL n=32), transthyretin (senile amyloid n=17, hereditary ATTR n=2), serum amyloid A (AA n=4), fibrinogen (AFib n=1), TGFb (corneal lattice amyloid n=1) and semenogelin (seminal vesicle amyloid n=1).Three diagnostically challenging cases are detailed as examples of the utility of LCM and tandem MS. The first case had extensive gastrointestinal amyloidosis and no evidence of clonal light chain disease; negative kappa, lambda, SAA and transthyretin immunohistochemistry; and negative genetic studies. Tandem MS revealed immunoglobulin lambda light chain type. The second diagnostically challenging case had: isolated renal amyloidosis with a positive AA stain and kappa restricted serum free light chains. Tandem MS revealed serum amyloid A2 protein. The third case had: cardiac, neurological and gastrointestinal involvement; and equivocal immunohistochemistry. Tandem MS demonstrated transthyretin and genetic studies showed a A97S ATTR mutation.Various other proteins were identified by tandem MS in amyloid extracts. Of particular interest is the presence of proteins typically known to be co-located in amyloid deposits which helps confirm that the microdissected tissue is amyloid. Typical amyloid-associated proteins were identified in the following number of cases: SAP (n=36), apolipoprotein A4 (n=42), vitronectin (n=44), apolipoprotein E (n=40) and clusterin (n=21). Various types of collagen were frequently present (n=29) and various, presumably contaminating, keratins were identified (n=24). ConclusionLCM and tandem MS allows correct typing of amyloid deposits in the majority of clinical biopsy samples. Disclosures:No relevant conflicts of interest to declare.

Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma.

Objective: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. Results: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. Conclusion: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute.

Mass Spectrometric Study of Stone Matrix Proteins of Human Bladder Stones

To evaluate the mechanisms of bladder uric acid stone (BUAS) formation by analyzing BUAS stone matrix proteins, with mass spectrometry (MS). Stone matrix proteins were extracted from 5 pure BUASs. The obtained proteins were analyzed with reverse phase liquid chromatography-tandem MS. The acquired data were investigated against a Swiss Prot human protein database, using Matrix Science Mascot. The identified proteins were submitted to UniProtKB website for gene ontology analysis to define their correlation. They were also submitted to Metacore platform and Kyoto Encyclopedia of Genes and Genomes website for pathway analysis. MS-determined protein expressions were validated by immunoblot. The liquid chromatography-tandem MS analysis identified 58-226 proteins in the 5 BUASs (450 proteins). Metacore software analysis suggests that inflammation might play an important role for BUAS formation. The analysis of endogenous metabolic pathways revealed that these proteins were categorized into glycerophospholipid or glycosphingolipid biosynthesis. Four of 5 identified proteins selected for validation, including uromodulin, S100P, Histone 4, and nucleophosmin, can be validated in the immunoblot data. Our results suggest that inflammatory process and lipid metabolism might play a role in the formation of BUAS. Whether these inflammatory responses are the etiology of stone formation or whether they result from local damage by stone irritation is uncertain.

AB0290 Serum protein biomarkers for response of rheumatoid arthritis to infliximab

BackgroundBiological therapies have been a great advance in Rheumatoid Arthritis (RA) treatment. However, up to a third of the RA patients do not respond to a given biologic and a...

Large pore dermal microdialysis and liquid chromatography‐tandem mass spectroscopy shotgun proteomic analysis: a feasibility study

The purpose of the present pilot study was to investigate the feasibility of combining large pore dermal microdialysis with shotgun proteomic analysis in human skin. Dialysate was recovered from human skin by 2000 kDa microdialysis membranes from one subject at three different phases of the study; trauma due to implantation of the dialysis device, a post implantation steady-state period, and after induction of vasodilatation and plasma extravasation. For shotgun proteomics, the proteins were extracted and digested with trypsin. Peptides were separated by capillary and nanoflow HPLC systems, followed by tandem mass spectrometry (MS/MS) on a Quadrupole-TOF hybrid instrument. The MS/MS spectra were merged and mapped to a human target protein database to achieve peptide identification and protein inference. Results showed variation in protein amounts and profiles for each of the different sampling phases. The total protein concentration was 1.7, 0.6, and 1.3 mg/mL during the three phases, respectively. A total of 158 different proteins were identified. Immunoglobulins and the major classes of plasma proteins, including proteases, coagulation factors, apolipoproteins, albumins, and complement factors, make up the major load of proteins in all three test conditions. Shotgun proteomics allowed the identification of more than 150 proteins in microdialysis samples from human skin. This highlights the opportunities of LC-MS/MS to study the complex molecular interactions in the skin.

GPU-Based Cloud Service for Smith-Waterman Algorithm Using Frequency Distance Filtration Scheme

As the conventional means of analyzing the similarity between a query sequence and database sequences, the Smith-Waterman algorithm is feasible for a database search owing to its high sensitivity. However, this algorithm is still quite time consuming. CUDA programming can improve computations efficiently by using the computational power of massive computing hardware as graphics processing units (GPUs). This work presents a novel Smith-Waterman algorithm with a frequency-based filtration method on GPUs rather than merely accelerating the comparisons yet expending computational resources to handle such unnecessary comparisons. A user friendly interface is also designed for potential cloud server applications with GPUs. Additionally, two data sets, H1N1 protein sequences (query sequence set) and human protein database (database set), are selected, followed by a comparison of CUDA-SW and CUDA-SW with the filtration method, referred to herein as CUDA-SWf. Experimental results indicate that reducing unnecessary sequence alignments can improve the computational time by up to 41%. Importantly, by using CUDA-SWf as a cloud service, this application can be accessed from any computing environment of a device with an Internet connection without time constraints.

“Zoom-In”—A Targeted Database Search for Identification of Glycation Modifications Analyzed by Untargeted Tandem Mass Spectrometry

Post-translational modifications (PTMs) are very important to biological function, however their identification and characterization is technically challenging. In this study, we have identified glycation modifications by nano LC-MSE, a data independent acquisition work flow, followed by database search using the Protein Lynx Global Server (PLGSJ). PLGS search with a complete human protein database hardly identified glycation modifications in a glycated human serum albumin (HSA), which was detected to be glycated by western blotting with advanced glycation end products (AGE) antibody and fluorescence spectroscopy. To overcome this difficulty, "Zoom-In" approach, a targeted database search was used to identify glycation modifications in a glycated HSA, which were further manually validated. This approach was useful for identification of glycation modifications from untargeted tandem mass spectrometryworkflow such as MSE, but may require the development of a new algorithm or an upgrade of the existing software.

G.P.57 Differential proteomic analysis of protein aggregates in desminopathy

Desminopathy is a subtype of myofibrillar myopathies (MFM) caused by mutations in DES, the gene encoding desmin. A histopathologic hallmark of the disease is a massive protein aggregation within skeletal muscle fibers. The aim of our study was to elucidate the composition of aggregates in MFM patients with different desmin mutations by using a label-free mass spectrometry approach. Aggregates and control tissue from muscle biopsies of MFM patients with four different mutations in DES were collected by laser microdissection and analyzed by a combination of mass-spectrometry and spectral index calculation. Proteins with a ratio >1.8 (sum of peptides identified in aggregates compared to intraindividual controls) were accepted as accumulated in aggregates. Results of selected proteins were validated by immunofluorescence studies. Mass spectrometric data were searched against an extended human protein database to detect desmin mutations at the protein level. Three hundred and seventeen different proteins were identified and 98 of them showed an accumulation in aggregates. Aggregate compositions were more heterogenous than in other MFM subtypes, depending on individual mutations, but desmin was on top of the list of abundant proteins in all cases except of one. Immunolocalization findings were consistent with proteomic data. Three out of four desmin mutations were identified at the protein level. Our proteomic approach enabled the identification of many novel components of pathologic protein aggregates within skeletal muscle fibers of desminopathy patients. This provides new insights in the pathogenesis of the disease. Differences in aggregate composition in patients with different desmin mutations indicate diverse pathomechanisms, consistent with data of previous functional studies.

A Highly Efficient Multifunctional Tandem Affinity Purification Approach Applicable to Diverse Organisms

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

Myosin heavy chain isoform expression in the Vastus Lateralis muscle of aging African green vervet monkeys

Non-human primates (NHP) represent an emerging animal model for the study of physical function, and provide opportunities for exploration of relationships of muscle biomolecular changes with age. One such primate model, the African green vervet monkey, has been used extensively in biomedical research but little is known regarding skeletal muscle composition, expression of myosin heavy chain (MHC) isoforms, and changes with age. In the present study we examined the effects of age on vastus lateralis (VL) muscle fiber-type composition, fiber cross-sectional area (CSA), and MHC isoforms expressed in 4 young and 4 older adult vervet monkeys. Proteomics analysis, using a human and nonhuman primate protein database, showed five MHC isoforms (I, IIA, IIX, IIB, and IIB′) expressed in female vervet VL muscle, which matched the human MHC isoforms. Fast type II fibers predominated and no pure type IIB or IIB’ containing fibers were detected. Hybrid fibers containing IIB/IIB’ MHC decreased in the old vervets. The CSA of both type I and type II fibers was significantly smaller in older vervet while type IIA fibers showed the most severity of atrophy. The decrease of fast MHC and atrophy of muscle fiber with aging recapitulate observations in human VL muscle. These findings, along with its homology of MHC between the vervet and human suggested that the vervet monkey may be a suitable preclinical model for understanding the cellular and molecular basis of sarcopenia and for developing new interventions to ameliorate the impact of disorders that affect skeletal muscle structure and function.

Mapping Ultra-weak Protein-Protein Interactions between Heme Transporters of Staphylococcus aureus

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.