Functional specifications of an integrated proteomics information management and analysis platform.

Detecting proteins in human blood holds the promise of a revolution in cancer diagnosis. Also, the ability to perform laboratory operations on small scales using miniaturized (lab-on-a-chip) devices has many benefits. Designing and fabricating such systems is extremely challenging, but physicists and engineers are beginning to construct such highly integrated and compact labs on chips with exciting functionality. This paper focuses on the presentation of the requirements of the information technology layer in such an integrated platform been developed in the LOCCANDIA project. LOCCANDIA is a Specific Targeted Research project (STREP) funded under the 6th Framework program of the EC. Its ultimate objective is to develop an innovative nano-technology based (lab-on-a-chip) platform for the medical-proeomics field. The paper presents the main engineering aspects, challenges and architecture for creating an Integrated Clinico-Proteomic Environment. The environment will be used to monitor and document the analysis and discovery chain and to allow the physician to interpret the digital spectrogram data delivered by the mass spectrometer, for diagnostic purposes.

New perspectives in the Escherichia coli proteome investigation.

Escherichia coli is a model organism for biochemical and biological studies as it is one of the best characterised prokaryote. Two-dimensional polyacrylamide gel electrophoresis, computer image analysis and different protein identification techniques gave rise, in 1995, to the Escherichia coli SWISS-2D PAGE database (http://www.expasy.ch/ch2d/). In the E. coli 3.5-10 SWISS-2D PAGE map, 40% of the E. coli proteome was displayed. The present study demonstrated that the use of narrow range pH gradients is able to potentially display up to a few copies of protein per E. coli cell. Moreover, the six new E. coli SWISS-2D PAGE maps (pH 4-5, 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11) presented here displayed altogether more than 70% of the entire E. coli proteome.

The mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and obesity.

A number of two-dimensional electrophoresis (2-DE) reference maps from mouse samples have been established and could be accessed through the internet. An up-to-date list can be found in WORLD-2D PAGE (http://www.expasy.ch/ch2d/2d- index.html), an index of 2-DE databases and services. None of them were established from mouse white and brown adipose tissues, pancreatic islets, liver nuclei and skeletal muscle. This publication describes the mouse SWISS-2D PAGE database. Proteins present in samples of mouse (C57BI/6J) liver, liver nuclei, muscle, white and brown adipose tissue and pancreatic islets are assembled and described in an accessible uniform format. SWISS-2D PAGE can be accessed through the World Wide Web (WWW) network on the ExPASy molecular biology server (http://www.expasy.ch/ ch2d/).

Heat shock proteins in human cancer.

The heat shock proteins (hsp) are ubiquitous molecules induced in cells exposed to sublethal heat shock, present in all living cells, and highly conserved during evolution. Their function is to protect cells from environmental stress damage by binding to partially denatured proteins, dissociating protein aggregates, to regulate the correct folding, and to cooperate in transporting newly synthesized polypeptides to the target organelles. The molecular chaperones are involved in numerous diseases, including cancer, revealing changes of expression. In this review, we mainly describe the relationship of hsp expression with human cancer, and discuss what is known about their post-translational modifications according to malignancies.

The 1999 SWISS-2DPAGE database update.

SWISS-2DPAGE (http://www.expasy.ch/ch2d/ ) is an annotated two-dimensional polyacrylamide gel electro-phoresis (2-DE) database established in 1993. The current release contains 24 reference maps from human and mouse biological samples, as well as from Saccharomyces cerevisiae, Escherichia coli and Dictyostelium discoideum origin. These reference maps have now 2824 identified spots, corresponding to 614 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence or any user-entered amino acids sequence. Last year improvements in the SWISS-2DPAGE database are as follows: three new maps have been created and several others have been updated; cross-references to newly built federated 2-DE databases have been added; new functions to access the data have been provided through the ExPASy proteomics server.

Modeling peptide mass fingerprinting data using the atomic composition of peptides.

The peptide mass fingerprinting technique is commonly used for identifying proteins analyzed by mass spectrometry (MS) after enzymatic digestion. Our goal is to build a theoretical model that predicts the mass spectra of such digestion products in order to improve the identification and characterization of proteins using this technique. We present here the first step towards a full MS model. We have modeled MS spectra using the atomic composition of peptides and evaluated the influence that this composition may have on the MS signals. Peptides deduced from the SWISS-PROT protein sequence database were used for the calculation. To validate the model, the variability of the peptide mass distribution in SWISS-PROT was compared to two theoretical, randomly generated databases. Functions have been built that describe the behavior of the isotopic distribution according to the mass of peptides. The variability of these functions was analyzed. In particular, the influence of sulfur was studied. This work, while representing only a first step in the construction of an MS model, yields immediate practical results, as the new isotopic distribution model significantly improves peak detection in MS spectra used by protein identification algorithms.

Improving protein identification from peptide mass fingerprinting through a parameterized multi-level scoring algorithm and an optimized peak detection.

We have developed a new algorithm to identify proteins by means of peptide mass fingerprinting. Starting from the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectra and environmental data such as species, isoelectric point and molecular weight, as well as chemical modifications or number of missed cleavages of a protein, the program performs a fully automated identification of the protein. The first step is a peak detection algorithm, which allows precise and fast determination of peptide masses, even if the peaks are of low intensity or they overlap. In the second step the masses and environmental data are used by the identification algorithm to search in protein sequence databases (SWISS-PROT and/or TrEMBL) for protein entries that match the input data. Consequently, a list of candidate proteins is selected from the database, and a score calculation provides a ranking according to the quality of the match. To define the most discriminating scoring calculation we analyzed the respective role of each parameter in two directions. The first one is based on filtering and exploratory effects, while the second direction focuses on the levels where the parameters intervene in the identification process. Thus, according to our analysis, all input parameters contribute to the score, however with different weights. Since it is difficult to estimate the weights in advance, they have been computed with a generic algorithm, using a training set of 91 protein spectra with their environmental data. We tested the resulting scoring calculation on a test set of ten proteins and compared the identification results with those of other peptide mass fingerprinting programs.

Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot.

To increase the throughput of protein identification and characterization in proteome studies, we investigated three methods of performing protein digestion in parallel. The first, which we term "one-step digestion-transfer" (OSDT), is based on protein digestion during the transblotting process. It involves the use of membranes containing immobilized trypsin which are intercalated between the gel and a PVDF collecting membrane. During electrotransfer, some digestion of the transferred proteins occurs, although poorly for basic and/or high molecular weight proteins. The second method is based on "in-gel" digestion of all proteins in parallel and termed "parallel in-gel digestion" (PIGD) to denote this fact. The PIGD led to more efficient digestion of basic and high molecular weight proteins (> 40,000) but suffered from a major drawback: loss of resolution for low molecular weight polypeptides (< 60,000) through diffusion during the digestion process. The third method examined was the combination of PIGD and OSDT procedures. This combination, called "double parallel digestion" (DPD), led to greatly improved digestion of high molecular weight and basic proteins without losses of low molecular weight polypeptides. Peptides liberated during transblotting of proteins through the immobilized trypsin membrane were trapped on a PVDF membrane and identified by mass spectrometry in scanning mode.

A molecular scanner to automate proteomic research and to display proteome images.

Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annoated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html + ++), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Method for identification and quantitative analysis of protein lysine methylation using matrix-assisted laser desorption/ionization--time-of-flight mass spectrometry and amino acid analysis.

Protein methylation is a post-translational modification that might have important functional roles in cell regulation. We present a new technique with sufficient sensitivity (sub-pmol level) for analysis of methylation of proteins in abundances typically found on proteome maps produced by two-dimensional (2-D) gel electrophoresis. The method involves the identification and quantitation of lysine (Lys) methylation using Fmoc (9-fluorenylmethyl chloroformate)-based amino acid analysis (AAA). Tri- and monomethyl-Lys were baseline-separated from other amino acids using a modified buffer system. Trimethyl-Lys was quantitatively recovered after acid hydrolysis and AAA of two known methylated proteins - yeast cytochome c and human calmodulin. The methylated peptides from tryptic digestion of those two proteins were identified by high sensitivity matrix-assisted laser desorption/ionization - time-of-flight (MALDI-TOF) mass spectrometry (MS). An automated mass-screening approach is proposed for the study of various post-translational modifications to understand the distribution of those protein isoforms separated by two-dimensional polyacrylamide gel electrophoresis. It is concluded that the combination of AAA and MALDI-TOF-MS provides a high sensitivity quantitative tool for the analysis of protein post-translational methylation in the context of proteome studies.

Structural characterization of Cu(I) and Zn(II) sites in neuronal-growth-inhibitory factor by extended X-ray absorption fine structure (EXAFS).

Neuronal-growth-inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approximately seven metal atoms/protein molecule, and 70% sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal composition, viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K-edge X-ray absorption spectra have been recorded for native Cu, Zn-GIF, Zn-substituted GIF, and these metals bound to the 32-residue N-terminal domain, Cu4-, Cu6- or Zn3-GIF-(1-32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu-S distance is approximately 2.25 A and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu...Cu backscattering is observed at approximately 2.67 A, indicative of the formation of Cu(x)(Scys)y clusters. Thus, the Cu(I) environment is similar to that observed in MT. This is also the case for Zn(II), with 4 S at approximately 2.34 A. However, in contrast to Zn-MT for Zn-substituted GIF and Zn3-GIF-(1-32), Zn...Zn backscattering is observed at approximately 3.28 A. The significance of these results are discussed with respect to the specific biological activity of GIF.