Revealing the amino acid composition of proteins within an expanded genetic code.

The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes.

YPED: a web-accessible database system for protein expression analysis.

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.

Probabilistic enrichment of phosphopeptides by their mass defect.

The mass defect, that is, the difference between the nominal and actual monoisotopic masses, of a phosphorus in a phosphate group is greater than for most other atoms present in proteins. When the mass defects of tryptic peptides derived from the human proteome are plotted against their masses, phosphopeptides tend to fall off the regression line. By calculating the masses of all potential tryptic peptides from the human proteome, we show that regions of higher phosphorylation probability exist on such a plot. We developed a transformation function to estimate the mass defect of a peptide from its monoisotopic mass and empirically defined a simple formula for a user-selectable discriminant line that categorizes a peptide mass according to its probability of being phosphorylated. Our method performs similarly well on phosphopeptides derived from a database of experimentally validated phosphoproteins. The method is relatively insensitive to mass measurement error of up to 20 ppm. The approach can be used with a tandem mass spectrometer in real time to rapidly select and rank order the possible phosphopeptides from a mixture of unmodified peptides for subsequent phosphorylation site mapping and peptide sequence analysis.

YPED: a proteomics database for protein expression analysis.

We have developed the Yale Protein Expression Database (YPED) to address the storage, retrieval, and integrated analysis of proteomics data generated by Yale's Keck Protein Chemistry and Mass Spectrometry Facility. YPED is Web-accessible and currently handles sample requisition, result reporting and sample comparison for ICAT, DIGE and MUDPIT samples. Sample descriptions are compatible with the evolving MIAPE standards. Peptides and proteins identified using Sequest or Mascot are validated with the Trans-Proteomic Pipeline developed at the Institute of Systems Biology and data from the resulting XML file are stored in the database. Researchers can view, subset and download their data through a secure Web interface.

Exploring the portability of informatics capabilities from a clinical application to a bioscience application.

This report describes XDesc (eXperiment Description), a pilot project that serves as a case study exploring the degree to which an informatics capability developed in a clinical application can be ported for use in the biosciences. In particular, XDesc uses the Entity-Attribute-Value database implementation (including a great deal of metadata-based functionality) developed in TrialDB, a clinical research database, for use in describing the samples used in microarray experiments stored in the Yale Microarray Database (YMD). XDesc was linked successfully to both TrialDB and YMD, and was used to describe the data in three different microarray research projects involving Drosophila. In the process, a number of new desirable capabilities were identified in the bioscience domain. These were implemented on a pilot basis in XDesc, and subsequently "folded back" into TrialDB itself, enhancing its capabilities for dealing with clinical data. This case study provides a concrete example of how informatics research and development in clinical and bioscience domains has the potential for synergy and for cross-fertilization.

The integration of similar clinical research data collection instruments.

We devised an algorithm for integrating similar clinical research data collection instruments to create a common measurement instrument. We tested this algorithm using questions from several similar surveys. We encountered differing levels of granularity among questions and responses across surveys resulting in either the loss of granularity or data. This algorithm may make survey integration more systematic and efficient.

Exploring issues of quality of service in a Next Generation Internet testbed: a case study using PathMaster.

This case study describes a project that explores issues of quality of service (QoS) relevant to the next-generation Internet (NGI), using the PathMaster application in a testbed environment. PathMaster is a prototype computer system that analyzes digitized cell images from cytology specimens and compares those images against an image database, returning a ranked set of "similar" cell images from the database. To perform NGI testbed evaluations, we used a cluster of nine parallel computation workstations configured as three subclusters using Cisco routers. This architecture provides a local "simulated Internet" in which we explored the following QoS strategies: (1) first-in-first-out queuing, (2) priority queuing, (3) weighted fair queuing, (4) weighted random early detection, and (5) traffic shaping. The study describes the results of using these strategies with a distributed version of the PathMaster system in the presence of different amounts of competing network traffic and discusses certain of the issues that arise. The goal of the study is to help introduce NGI QoS issues to the Medical Informatics community and to use the PathMaster NGI testbed to illustrate concretely certain of the QoS issues that arise.