Christopher Colangelo

The Proteomics Standards Research Group (sPRG) is reporting the first year progress in a two-year sPRG 2012-2013 study which focuses on the generation of a standard that can be used for interassay, interspecies, and interlaboratory normalization in both label-free and stable isotope label-based quantitative proteomics analysis. The standard has been formulated as two mixtures: 1000 stable isotope 13C/15N-labeled synthetic tryptic peptides alone, and peptides mixed with a tryptic digest from a HEK 293 cell lysate. The sequences of the synthetic peptides were derived from approximately 400 proteins and were conserved across proteomes of the most commonly analyzed species: Homo sapiens, Mus musculus and Rattus norvegicus. The selected peptides represent the full range of hydrophobicities and isoelectric points typical to tryptic peptides from complex proteomic samples. The standard was designed to represent proteins of various concentrations, spanning three orders of magnitude. This year we focused our efforts on selection of appropriate protein and peptide candidates, peptide synthesis, quality assessment and LC-MS evaluation by several sPRG member laboratories. The sPRG study design and initial results of a thorough characterization of the standard using a variety of instrumental configurations and bioinformatics approaches will be presented in this talk.The sPRG is hopeful that the designed formulation will become a valuable resource in various mass spectrometry-based proteomic applications, including quantitative and differential profiling, as well as general benchmarking (e.g. chromatographic retention time). The sPRG plans to start recruiting study participants in April 2013, complete the study by the end of the year 2013, and present the results at the ABRF 2014 meeting. The sPRG encourages proteomics laboratories to participate in the study and sign in at www.abrf.org/sprg.The second half of the session will discuss the qualitative stability study performed using purified synthetic peptides containing a variety of modifications selected from the 2012 sPRG ABRF sample. The stability of the selected synthetic peptides was evaluated by the sPRG using different storage conditions over a three-month period. After storage at either at room temperature, +4°C or −80°C for one week, one month, or three months. Quantitative LC-MS/MS analysis was used to monitor the stability and degradation of the peptides, and to determine the effect of modifications and storage conditions on peptide degradation rates. The data presented have been built on the quantitative study that was presented at both the 2012 ABRF and ASMS conferences. All forms of degraded peptides were separated and identified using nano-LC tandem mass spectrometry on a Thermo Scientific Q-Exactive hybrid mass spectrometer. Integrated extracted ion chromatograms were used to measure relative amounts of degradation to identify which pathways are most prevalent during storage.

Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. Potential biomarkers were identified by using a multi-proteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope-labeled internal peptide standards were synthesized for scheduled multiple reaction monitoring (MRM). The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least 2 transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from amongst 167 urinary proteins. This appro...

Transmissible encephalopathies (TSEs), such as Creutzfeldt-Jakob disease (CJD) and scrapie, are caused by infectious agents that provoke strain-specific patterns of disease. Misfolded host prion protein (PrP-res amyloid) is believed to be the causal infectious agent. However, particles that are stripped of PrP retain both high infectivity and viral proteins not detectable in uninfected mouse controls. We here detail host proteins bound with FU-CJD agent infectious brain particles by proteomic analysis. More than 98 proteins were differentially regulated, and 56 FU-CJD exclusive proteins were revealed after PrP, GFAP, C1q, ApoE, and other late pathologic response proteins were removed. Stripped FU-CJD particles revealed HSC70 (144× the uninfected control), cyclophilin B, an FU-CJD exclusive protein required by many viruses, and early endosome-membrane pathways known to facilitate viral processing, replication, and spread. Synaptosomal elements including synapsin-2 (at 33×) and AP180 (a major FU-CJD exclusive protein) paralleled the known ultrastructural location of 25 nm virus-like TSE particles and infectivity in synapses. Proteins without apparent viral or neurodegenerative links (copine-3), and others involved in viral-induced protein misfolding and aggregation, were also identified. Human sCJD brain particles contained 146 exclusive proteins, and heat shock, synaptic, and viral pathways were again prominent, in addition to Alzheimer, Parkinson, and Huntington aggregation proteins. Host proteins that bind TSE infectious particles can prevent host immune recognition and contribute to prolonged cross-species transmissions (the species barrier). Our infectious particle strategy, which reduces background sequences by >99%, emphasizes host targets for new therapeutic initiatives. Such therapies can simultaneously subvert common pathways of neurodegeneration.

Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated. To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation. We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and up to 72 hours after transplantation revealed that 22 proteins including haptoglobin, a known anti-oxidant, are significantly upregulated in our grafts. Through the use of haptoglobin deficient mice, we show that 80% of haptoglobin deficient recipients treated with peri-operative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited > 100 days survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild type recipients rejected their transplants by 21 days po...

Stable changes in neuronal gene expression have been studied as mediators of addicted states. Of particular interest is the transcription factor ΔFosB, a truncated and stable FosB gene product whose expression in nucleus accumbens (NAc), a key reward region, is induced by chronic exposure to virtually all drugs of abuse and regulates their psychomotor and rewarding effects. Phosphorylation at Ser(27) contributes to ΔFosB's stability and accumulation following repeated exposure to drugs, and our recent work demonstrates that the protein kinase CaMKIIα phosphorylates ΔFosB at Ser(27) and regulates its stability in vivo. Here, we identify two additional sites on ΔFosB that are phosphorylated in vitro by CaMKIIα, Thr(149) and Thr(180), and demonstrate their regulation in vivo by chronic cocaine. We show that phosphomimetic mutation of Thr(149) (T149D) dramatically increases AP-1 transcriptional activity while alanine mutation does not affect transcriptional activity when compared wi...

Attempts to correlate protein abundance with mRNA expression levels have had variable success. We review the results of these comparisons, focusing on yeast. In the process, we survey experimental techniques for determining protein abundance, principally two-dimensional gel electrophoresis and mass-spectrometry. We also merge many of the available yeast protein-abundance datasets, using the resulting larger 'meta-dataset' to find correlations between protein and mRNA expression, both globally and within smaller categories.

We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC-MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay.

The rapidly growing field of proteomics seeks to track changes in protein expression function that underlie the growth and differentiation of individual cell types, both during normal development and during the onset and progression of disease. Recent years have seen great strides in mRNA expression analysis, and the development of new technologies for protein profiling. However, current methods are limited to analysis of the relative expression level of only a few hundred to perhaps 2000 proteins, well below the ability of DNA microarrays to potentially interrogate the mRNA expression of more than 25,000 genes. Proteomics faces a special challenge in studies of the nervous system, where cellular and sub-cellular architecture is among the most complex in the body. This article presents an overview of current proteomic profiling technologies, reviews the recent use of some of these approaches in studies of the nervous system, and discusses the potential application of neuroproteomics to studies of drug addiction.

Selective or Multiple Reaction monitoring (SRM/MRM) is a liquid-chromatography (LC)/tandem-mass spectrometry (MS/MS) method that enables the quantitation of specific proteins in a sample by analyzing precursor ions and the fragment ions of their selected tryptic peptides. Instrumentation software has advanced to the point that thousands of transitions (pairs of primary and secondary m/z values) can be measured in a triple quadrupole instrument coupled to an LC, by a well-designed scheduling and selection of m/z windows. The design of a good MRM assay relies on the availability of peptide spectra from previous discovery-phase LC-MS/MS studies. The tedious aspect of manually developing and processing MRM assays involving thousands of transitions has spurred to development of software tools to automate this process. Software packages have been developed for project management, assay development, assay validation, data export, peak integration, quality assessment, and biostatistical analysis. No single tool provides a complete end-to-end solution, thus this article reviews the current state and discusses future directions of these software tools in order to enable researchers to combine these tools for a comprehensive targeted proteomics workflow.

Panorama is a web application for storing, sharing, analyzing, and reusing targeted assays created and refined with Skyline,1 an increasingly popular Windows client software tool for targeted proteomics experiments. Panorama allows laboratories to store and organize curated results contained in Skyline documents with fine-grained permissions, which facilitates distributed collaboration and secure sharing of published and unpublished data via a web-browser interface. It is fully integrated with the Skyline workflow and supports publishing a document directly to a Panorama server from the Skyline user interface. Panorama captures the complete Skyline document information content in a relational database schema. Curated results published to Panorama can be aggregated and exported as chromatogram libraries. These libraries can be used in Skyline to pick optimal targets in new experiments and to validate peak identification of target peptides. Panorama is open-source and freely available...

X-ray absorption spectroscopy has been used to define the copper-site structures in both the resting (oxidized) and dithionite-reduced states of amine oxidases from bovine plasma, porcine plasma, porcine kidney, pea seedlings, and the gram-positive bacterium Arthrobacter P1. ...

... We are indebted to Martin J. George of SSRL for use of his data collection software and to Richard Glass and John Enemark, both of the University of Arizona, for helpful comments. ... VN; Khangulov, SV; Axley, MJ; Stadtman, TC Proc. Natl. Acad. Sci. USA 1994, 91, 7708−7711. ...

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies.

We have developed an integrated web-accessible software system called the Yale Protein Expression Database (YPED) to address the need for storage, retrieval, and integrated analysis of large amounts of data from high throughput proteomic technologies. YPED is an open source system which integrates gel analysis results with protein identifications from DIGE experiments. The system associates the DIGE gel spots and image, analyzed with DeCyder, with mass spectrometric protein identifications from selected gel spots. Following in gel trypsin digestion, proteins in spots of interest are analyzed using MALDI-TOF/TOF on an AB 4700 or, more recently, on an AB 4800 with protein identifications performed by Mascot in conjunction with the AB GPS Explorer system. In addition to DIGE, YPED currently handles protein identifications from MudPIT, iTRAQ, and ICAT experiments. Sample descriptions are compatible with the evolving MIAPE standards. Tandem MS/MS results from MudPIT, and ICAT analyses are validated with the Trans-Proteomic Pipeline and then stored in the database for viewing and linking to the identified proteins. Researchers can view, subset, and download their data through a secure Web interface that includes a table containing proteins identified, a sample summary, the sample description, and a clickable gel image for DIGE samples. Tools are available to facilitate sample comparison and the viewing of phosphoproteins. A summary report with PANTHER Classification System annotations is also available to aid in biological interpretation of the results. The source code is open-source and is available from http://yped.med.yale.edu/yped_dist.

The gene for an archaeal homolog of the eukaryotic transcription factor TFIIB has been cloned from the marine hyperthermophilic archaeon Pyrococcus furiosus and overexpressed in Escherichia coli. This TFB gene displays a sequence that is identical to a gene sequence in P. woesei. A gene for the 49-residue N-terminal domain of TFB that contains a putative C-X2-C-X15-C-X2-C metal-binding motif was subcloned and overexpressed as TFB-NTD. Purification of the TFB-NTD gene product yields Zn- and Fe-containing forms, which have been characterized by mass spectrometry and UV-visible, electron paramagnetic resonance, and X-ray absorption (XAS) spectroscopies. Only the Zn form of the TFB holoprotein has been (partially) purified, and it has been characterized by XAS. All spectroscopic characteristics are consistent with a nearly tetrahedral MS4 metal-binding site made up of the four cysteine residues in the N-terminal domain. The relatively greater thermal stability of the Zn form suggests that TFB may be a Zn-containing protein involved in archaeal transcription.