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Over the last ten years the Proteomics Research Group (PRG) has undertaken technical studies that have covered a wide range of issues unique to the rapidly developing field of proteomics and have included a range of qualitative and quantitative experiments. The PRG studies have resulted in a great deal of attention not only within the ABRF community but also outside as is evident from numerous articles dealing with proteomics methods, procedures and standardization. As the field continues to develop, the diversity of instrumentation and laboratory workflows have grown in tandem. Therefore, in the PRG2011 study it seemed especially useful to perform a survey to help the PRG define future studies based on the current blend of sample types and technologies and obtain a view of emerging trends. A survey was created to ascertain three main insights into core facility function: 1) How labs interact with their clients, 2) The capacity of labs to meet the demands of their clients, and 3) The blend of experimental techniques offered to and requested by clients. Survey questions were designed to obtain information from both users of core facilities and the directors and personnel of core facilities. Questions covered such topics as the type and age of instruments in use, how data is analyzed and presented to client, sources of funding, and emerging proteomics trends. Results are compiled en masse and presented without regard to institution. Early results reveal that about 2/3 of the responders are not ABRF members, and at least one lab still has an operational mass spectrometer that was acquired in 1990!

The ABRF Metabolomics Research Group (MRG) was formed in 2009 and aims to educate research scientists and resource facilities in the analytical approaches and management of data resulting from comprehensive metabolite studies and to promote the science and standardization of metabolomic analyses for a variety of applications. Last year the MRG conducted a ‘Survey Study’ on the current use of metabolomics technologies and procedures in core facilities. This year the MRG is organizing a ‘Research Study’ involving a spiked plasma sample. The study sample consists of a human biofluid as the matrix, replicating a typical small scale metabolomics pilot experiment that either a core or research laboratory would perform. The sample consists of two groups of normal human plasma (NIST plasma ‘Standard Reference Material’) with spiked in compounds. There are three biological replicates in each group (n = 3 design) with different levels of spiked compounds differentiating the two groups. Participants are asked to determine statistical significance, fold change, and identify compounds that differ significantly between groups A and B. The design reflects issues encountered in an actual metabolomics experiment conducted with human or animal specimens. The study is compatible with many methodological approaches in metabolomics, including, but not limited to LC/MS, GC/MS, NMR, as well as other methods. As with any metabolomics profiling experiment, the best results would be expected using a combination of approaches. The study is the first of its kind in the field of metabolomics and is expected to produce important information on the strengths and limitations of the various platforms and technologies that are commonly used for comprehensive metabolite analyses.

Characterizing the assembly of multi-protein complexes and the competition between multiple protein ligands for a given target are common challenges faced by core facilities. The MIRG2010 Benchmark study was designed to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies such as surface Plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B and C) and asked to determine if a ternary A-B-C complex can form, or if ligands B and C bind competitively to protein A. This presentation will summarize the conclusions from the 2010 Benchmark Study, and provide perspective on the potential for future application of this system as a reference standard for quantitative characterization of protein-protein interactions using biosensor technologies. The field of label-free biophysical technologies like surface plasmon resonance (SPR) and isothermal calorimetry (ITC) are becoming indispensable in translational research and in the discovery phase of biotherapeutics. Investigators are much more aware about the developments in biomolecular interaction analysis using SPR and ITC and usefulness of these technologies in designing better drugs based on biomolecules and vaccines. The Molecular Interaction Research Group (MIRG) of ABRF has conducted an on-line survey to capture the recent explosive developments in these technologies. The survey was targeted to both academia and pharmaceutical industry and the survey data will be presented during the meeting.

Metagenomics is one of several genomics applications, which has benefited immensely from the high throughput and cost efficacy of Next generation sequencers. And although hundreds of studies on metagenome analysis have been published over the past few years, the methodology for conducting them is still very much evolving. In this DSRG study we will evaluate the influence of various sample preparation methods, specifically DNA extraction and amplification approaches, on data output along with a comparative analysis of Next generation sequencing platforms. We will study the effect of these different experimental and technical strategies on determination of sample biodiversity.

The evolution of genomic technologies is occurring rapidly and often requires large amounts of source DNA. There is also an expanded desire to analyze smaller numbers of cells for higher resolution studies as well as to take advantage of large numbers of archived samples (e.g. FFPE, serum, etc.). To provide enough material for the newest genomic technologies, whole genome amplification (WGA) has reemerged as an important and necessary technique. With some new WGA products on the market, we have evaluated the quantity and quality of WGA products generated as well their performance on some of the current genomic applications. The GVRG has completed a benchmarking study evaluation of 6 commercially available WGA kits using several genotyping assays. Utilizing 6 samples, the different WGA kits were tested following the manufacturer's protocols. The samples included Coriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, both from the same individual (1 newly extracted and the other extracted 2 years ago), and 1 sample from the Coriell trio that was fragmented prior to amplification for a total of 6 samples tested. The WGA kits included those from Sigma, NuGen, Qiagen and GE Life Sciences. The 6 samples were amplified as blind duplicates at several GVRG member lab sites resulting in a total of 78 amplified products. Quality metrics were performed on the 78 amplified products using several different techniques in order to provide measurements on yield, fragment size, and concentration. Several widely used genotyping methods were used to evaluate the amplification products, including Illumina Human Omni1 Quad Beadchip, TaqMan copy number and SNP genotyping assays, and STR genotyping. Data will be presented on genotyping concordance and loss of heterozygosity checks across the different platforms where possible.

The Nucleic Acid Research Group (NARG) has previously conducted studies evaluating the impact of RNA integrity and priming strategies on cDNA synthesis and real-time RT-qPCR. The results of last year's field study as it relates to degraded RNA will be presented. In continuation of the RNA integrity theme, this year's study was designed to evaluate the impact of RNA integrity on the analysis of miRNA expression using real-time RT-qPCR. Target section was based on data obtained by the Microarray Research Group (MARG) and other published data from next gen sequencing. These 9 miRNAs represent three groups of miRNA that are expressed at low, medium or high levels in the First Choice human brain reference RNA sample. Two popular RT priming strategies tested in this study include the Megaplex miRNA TaqMan assay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences). The basis for the ABI assay design is a target-specific stem-loop structure and reverse-transcription primer, while the Qiagen design combines poly(A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study, the human brain reference RNA was subject to controlled degradation using RNase A to RIN (RNA Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded).These templates were then used to assess both RT methods. In addition to this real-time RT-qPCR data, the same RNA templates were further analyzed using universal poly(A) tailing and hybridization to Affymetrix miRNA GeneChips. This talk will provide insights into RT priming strategies for miRNA and contrast the qPCR results obtained using different technologies.

Deconvolution is a computational technique used to remove blur from images. In this presentation image formation in the microscope will be reviewed and it will be explained what deconvolution does and why deconvolution is needed as a post acquisition processing step. The concept of Point Spread Function (PSF) will be introduced. Several approaches to deconvolution and deblurring exist from simple subtractive methods to complex iterative approaches. The major approaches will be examined. It will be explained why in the presence of noise a statistical approach is required. Blind deconvolution will be introduced. The concept of Point Spread Function will be examined in detail. The effect of microscope modality, lens parameters, and specimen parameters on PSF shape will be discussed. Theoretical PSF calculation and PSF measurement using beads will be reviewed with examples. It will be explained why theoretical and measured PSFs can differ from the true system PSF. Blind deconvolution will be further examined as a tool to deal with the uncertainty of the true form of the PSF. Practical guidelines for data acquisition will be reviewed. The relationship between sampling rate and quality of deconvolution results will be explained.

The Proteomics Standards Research Group (sPRG) has initiated a study that focuses on development of a standard that can be used in both assessment of a laboratory's ability to detect an array of post-translational modifications in a complex proteomic sample and development of new approaches for characterization of post-translationally modified proteins. The sample that has been generated for the first stage of this study contains a mixture of more than seventy synthetic peptides with a variety of modifications, in a tryptic digest of six proteins. Modifications represented in the sample include acetylation, methylation, nitration, phosphorylation, and sulfation. The individual proteins were purified, digested and analyzed prior to formulation of the sample. The synthetic peptides were each analyzed and then mixed with the digests; the mixture was aliquoted and lyophilized in sufficient quantities that would permit evaluation by several analytical approaches. The sample was fully characterized by members of the sPRG. It is planned that in 2011, the sample will be distributed to requesting laboratories for a larger study focusing on PTM characterization. The sPRG will present results obtained at different stages of preparation and characterization of the sample. Approaches for analysis of complex proteomic samples containing various post-translationally modified proteins will also be discussed. The sPRG is planning to open the study for sample requests at the ABRF 2011 conference. From the responses returned by the participating laboratories, the sPRG will subsequently report on the effectiveness of the approaches used for characterization of posttranslational modifications in protein digests.

Members of the MARG will discuss our research projects: Comparison of microarray and deep sequencing platforms for microRNA profiling, Performance of a synthetic human microRNA reference panel, Participation in the SEQC Sequencing Quality Control consortium, and RNA-Seq profiling of environmental samples exposed to the Gulf oil spill.

Modern light microscopes are highly evolved opto-electronicmechanical devices. Establishing instrument performance is crucial in ensuring that reliable and accurate images can be acquired. Imagers, as well as granting agencies, need to be confident that data collected will be uniform and quantifiable both temporally and from instrument to instrument. Last year, in phase-one of our world-wide research study on instrument performance, we successfully concentrated our efforts on three image-based tests: long and short term stability of illumination sources, uniformity of field illumination, and co-registration across various wavelengths. A manuscript summarizing the phase-one study has been submitted to and accepted by Microscopy & Microanalysis, one of the highest rated imaging journals. In the coming year, phase two of our instrument performance study will focus on determining the following: the point spread function of an imaging system, the system's spectral separation ability and the spectral calibration and resolution of the detection system. As with the phase-one study, the goal of this study will be neither to compare the performance of different brands of instruments, nor to ascertain which brand had better performance in a given area. Instead, the goal of our proposed phase-two study is continue to focus on determining the current state of modern imaging systems through straightforward, efficient and robust tests. These tests will aid imagers in the early detection of system problems. Moreover, these tests will continue to help define relative standards that will assist both core personnel and imagers in maintaining their instruments in optimal operating conditions.

Establishing the N-terminal sequence of intact proteins plays a critical role in biochemistry and potential drug development. N-terminal sequence analysis is necessary for quality control of protein biologics, for determining sites of signal peptide cleavage events, as a first step in elucidating the sequences of genes from uncommon species and for the characterization of monoclonal antibodies. Automated Edman degradation has been the method of choice for these analyses. However, alternate methods for N-terminal sequence analysis have emerged. The recent PSRG studies have established that Edman sequencing and mass spectrometry based techniques have varied strengths and weaknesses depending on several experimental factors and both play an important role in terminal sequencing. With this complimentary role realized, the 2011 PSRG study attempts to evaluate the sensitivity limits of the various sequencing techniques. The PSRG distributed three sample sets of 3 tubes each, varying by sample format (lyophilized, gel slice or membrane piece). Each set of three samples contains the same recombinant protein with increasing amounts of material. The sequence of this protein is not listed in any database. Participants could request any one, two, or all three sample sets. Including PSRG committee, a total of 38 participants requested 74 sample sets. The participants were asked to determine as many amino acids from both termini by their method of choice, and were encouraged to try multiple methods for sequence elucidation. Study participants were directed to a website to anonymously upload sequences and supporting data. Our analysis focuses on the length and accuracy of the sequence calls reported by the participants, and how that compares with decreasing amounts of protein and the type of sample format analyzed. A comparison of the results obtained by Edman chemistry and by alternative technologies as well as information on the type of instruments and protocols is reported.

The possibilities of personalized medicine are largely rooted in successfully exploiting the ever-growing genome and proteome-wide understanding of human biology. Translating this knowledge base into the clinic requires, among other things, being able measure the patient's genomic and proteomic disease signature in a routine manner. Thus, expanded focus on assays and platforms capable of measuring these specific signatures within tens, hundreds or even thousands of complex patient samples simultaneously is critical. Antibody-based assays and platforms have become a centerpiece of many technologies pushing these boundaries because of the antibody's intrinsic specificity, diverse recognition capacity and ease with which they can be produced. Furthermore, these same characteristics have made them an equally promising prospect for therapeutic approaches. This seminar trifecta will highlight the pivotal role antibodies have in multiplex biomarker assay development and therapeutic venues. Specifically: 1. Brian Curtis will discuss a robust multiplex assay platform to detect human platelet auto-antibodies and platelet antigen polymorphisms by combining bead array technologies with flow cytometry. Such a simple, yet high-throughput platform would facilitate routine testing for platelet disorders like neonatal alloimmune thrombocytopenia, multi-platelet transfusion refractoriness, and post-transfusion purpura; 2. Robert Umeck will summarize the challenges in developing antibody-based multiplex assays and how Meso Scale Discovery utilizes an electrochemiluminescence detection system combined with a patterned multi-array format to generate multiplex assays for nearly any application. Several actual examples will be presented to demonstrate how this approach could support personalized medicine; 3. John Harlan will introduce and discuss the evolution of therapeutic antibodies. To illustrate where the field is venturing, he will highlight Abbott Laboratory's new bi-specific antibody format.

The field has undertaken over the past few years a series of studies to evaluate methods for glycan analysis. There are several methods that are widely used at this time for characterization of glycoprotein glycans that appear to be generally reliable. These include (1) reductive amination followed by reversed phase chromatography, (2) reductive amination with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS), (3) reductive amination with electrospray liquid chromatography-MS, and (4) permethylation with tandem MS. The ABRF glycoprotein research group (gPRG) is planning a study in which participant laboratories carry out glycan analysis according to protocols representing best practice in the field. The results will be presented at the 2012 ABRF meeting. The results will be useful for demonstrating the reproducibility of glycan analysis using comparable protocols. They will also highlight the extent to which different methods bias the glycan analysis results obtained. This workshop will summarize the history and use of the glycan analysis methods proposed for the 2012 study. There will be opportunity for discussion and comment on these methods.

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Over the last four years, we witnessed the tremendous advances in Next Generation Sequencing (NGS) that have dramatically decreased the cost of whole genome sequencing. However, the cost of sequencing larger genomes is still significant. In addition and depending on the goal of study, whole genome sequencing creates a large amount of additional/auxiliary data that complicates data analysis. There are several commercial methods available for isolating subsets of genomes that greatly enhance the efficiency of NGS by allowing researchers to focus on their regions of interest. For the 2009–11 DSRG study, we compared products from two leading companies; Agilent and Nimblegen, that offer custom enrichment methods. Both companies obtained the same genomic DNA stock and performed DNA capture on the same specified regions. Following capture, the Illumina Genome Analyzer IIx system was used, in two different laboratories, to generate the sequence data. We present our data comparing in terms of cost, quality, reproducibility and most importantly completeness and depth of coverage. Acknowledgements: We would like to thank Agilent, Illumina and Nimblegen for all their support in making this study possible.

The field of mass spectrometry based proteomics has seen several key innovations over the last several years, including novel experimental methods, new instruments, and unique fragmentation strategies. The latter, in the form of electron capture dissociation (ECD) and electron transfer dissociation (ETD) have captured the imaginations of many researchers, expanding their ability to identify and analyze peptides and proteins. However, since ECD/ETD spectra differ substantial from more traditional collision induced dissociation (CID) spectra in both their prominent ion series as well as their preferred bond-breaking characteristics, the (automatic) interpretation of ECD/ETD spectra requires novel algorithm optimizations. Efficient identification of ECD/ETD spectra thus remains an active and exciting field of proteomics informatics research. In this work, the ABRF Proteome Informatics Research Group (iPRG) presents the results of a collaborative study focusing on the analysis of an LC-MS/MS dataset from a yeast lysate digested with Lys-C and enriched for highly charged peptides using strong cation exchange fractionation. The data derived from one fraction analyzed exclusively by ETD was distributed to participants for analysis in several equivalent formats, along with a standardized sequence database derived from the UniProtKB/Swiss-Prot yeast complement, a decoy version of this database, and an applicable spectral library. Participants were free to use any and all methods available to them to identify this fraction, and results were to be submitted using an Excel template. All participant identities were subsequently anonymized, and a survey was used to collect information about participant experience and software tools used to produce the submitted analysis. This uniform collection of data has allowed a thorough comparison of participant results. A summary, including a comparison of results submitted by members of the iPRG, will be presented.