References

Anderson NL, Taylor J, Scandora AE, Coulter BP, Anderson NG (1981) The TYCHO system for computer analysis of two-dimensional gel electrophoresis patterns. Clin Chem 27 1807-1820 Appel RD, Hochstrasser DF, Funk M, Vargas JR, Pellegrini C, Muller AF, Scherrer JR (1991) The MELANIE project from a biopsy to automatic protein map interpretation by computer. Electrophoresis 12 722-735 Appel RD, Palagi PM, Walther D, Vargas JR, Sanchez JC, Ravier F, Pasquali C, Hochstrasser DF (1997a) Melanie II - a...

Disease Diagnosis from Body Fluids

The use of proteomics of body fluids for the diagnosis and monitoring of disease is a difficult task. Many proteomic techniques have been applied for the profiling of body fluids, including 2-D electrophoresis (2-DE), surface-enhanced laser desorption ionisation (SELDI), isotope-coded affinity tag (ICAT) labelling and 2-D protein fractionation. However, the real impact of proteomics on disease diagnosis and patient care to date is insignificant. During the last 10 years, a very small number of...

Info

IAH FiTDLGE EHF glvlia fsqylqqcpf dehv lvnel tefa tcvad 81 whagce ,sL HTLFGDELO vasL etygd madcc qep s necflsh ddspdlp L FDPMTLCDEF ADE fug 161 ylyeia hp yfyapellyy an yngvfqe ccqaed qac lip ietm e vlaaaa q 1 casiq fg 6 al awsua 241 lsq fp ae lvtd It vh CCH gdllecadd adla yicdn qdtias .rE CCDKPLLE s hciaeve da 321 ipenlpplta dfeed dvc nyqea DAF L GSPLYEYSFF HPEYAVSVLL la ayeatl eecca ddph acyatvfd 1 101 kHLVDEPQNL IKQNCDQfEK LGBYGFQNAL IVRytrKVPQ VSTPTLVEVS Rolgkvgtrc Ct- pe3e mp ctedylslil...

Protein Identification by Mass Spectrometry

Silico Peptide

MS is now the technique of choice for protein identification. A number of approaches can be used and these are discussed next. 3.3.1 'Top-Down' Versus 'Bottom-Up' Strategies for Protein Identification When analysing proteins by MS, we can use a 'top-down' or a 'bottom-up' approach. In the 'top-down' approach, whole proteins are purified and fragmented in the mass spectrometer. This approach is usually restricted to the more powerful Fourier transform (FT) mass spectrometers that have the Fig....

Url

(no Web interface) as a general, high-throughput method. Alternatively, numerous groups have generated amino acid composition data by hydrolysing individual proteins from 2-DE gels with strong acid, and then analysing the resulting free amino acids with chromatographic techniques (Eckerskorn et al. 1988 Jungblut et al. 1992 Wilkins et al. 1996a). Amino acid composition based identification can then be performed with the AACompIdent tool (Table 3.1). Again, the search can be narrowed with other...

Analytical Aspects

The complexity of biological samples remains a key issue. As stated already, the tremendous chemical diversity of proteins and their enormous range of abundance are impediments to rapid progress in proteomics. The proteomic workflow requires massive parallel fractionation to analyse proteomes in more depth. It therefore remains a challenge to obtain reproducible, qualitative and quantitative, accurate and sensitive results. Further miniaturisation and automation should ultimately provide the...

Proteomics and Cancer

Cancer is a major public health problem worldwide and a leading cause of death in developed countries. Recently, collaborative work between the World Health Organization and the International Agency for Research on Cancer reported that more than ten million new cancers were diagnosed in 2000 and that there were over six million deaths from the disease (Stewart and Kleihues 2003). Nowadays, the incidence of cancer continues to increase slightly. However, the mortality rate from all cancers...

Pathogenesis of Type 2 Diabetes

Type 2 diabetes is characterised by an abnormal glucose homeostasis leading to hyperglycaemia. Diagnosis often occurs after many years of a prediabetic state accompanied by an absence of pathognomic symptoms. When diabetes is suspected, the standard test is to measure glucose concentration in the plasma after an overnight fast. The diagnostic value is considered to be around 7.1 mmol L and has to be measured on two different occasions. The oral glucose tolerance test or the intravenous glucose...

Introduction to Diabetes

The identification of new molecular targets that could lead to new treatments for diabetes is an area where proteomics could have a key role. Epidemiological data from the late nineteenth century described diabetes mellitus (from the Greek for 'pass through' and the Latin for 'sweet as honey') as a rather frequent disorder in man, in obese people above 50 years old, in cities and in western countries (Pavy 1894). Even at that time, diabetes was seen as a disease of urban life. There are two...

Swiss2dpage

SWISS-2DPAGE is an annotated database that assembles data on proteins from a variety of human and mouse biological samples as well as from Arabidopsis thaliana, Dictyostelium discoideum, Escherichia coli, Saccharomyces cerevisiae and Staphylococcus aureus. In all cases, proteins have been identified on 2-D polyacrylamide gel electrophoresis (PAGE) reference maps. SWISS-2DPAGE provides links between sequence data and protein expression. Most recorded proteins have been identified by one or more...

Some Interesting Modification Stories

The literature is just beginning to better describe the importance of post-translational modifications. To illustrate this concept, we have chosen some 'stories' of specific proteins whose activity is affected by one or more modifications to the expressed amino acid sequence. Our focus is on some of those which are known to be important to humans in a medical context. Erythropoietin has become the most produced recombinant protein drug in the world. It increases patient red blood cell counts...

Imaging Mass Spectrometry

Medical imaging techniques, such as computer tomography and magnetic resonance imaging, play an important role in the study of anatomical, physiological and functional information however, these images do not display protein distributions. Histochemical methods provide 2-D maps of protein distribution by way of labels, affinity tags or expression markers, but they are limited to the simultaneous analysis of only a few known proteins at a time. An alternative method has been developed by...

Liquid Chromatography Mass Spectrometry

The intensive use of MS in proteomics has opened a new domain in proteome imaging. The representation of LC-MS datasets as a 2-D plot can highlight nuances of data not necessarily seen when displayed as 1-D chromatographs or spectra. In a LC-MS image, the axes represent the retention time derived from LC and the mass-to-charge (m z) values derived from MS, while the grey levels of the image describe the MS signal intensities. The idea of displaying LC-MS data as an image is quite recent (Berger...

Conventional Sample Preparation

Sample preparation for isoelectric focusing (IEF) relies on non-ionic or zwit-terionic reagents to disrupt protein complexes and denature proteins. This ensures that only polypeptide monomers are subjected to the subsequent electrophoretic separation. Because IEF separates proteins on the basis of isoelectric point, the single most powerful solubilising reagent, the anionic surfactant sodium dodecyl sulfate (SDS), is not normally used as it strongly attaches to proteins and may cause anomalous...

Cy3Cy5

Fig. 4.3 Structures of the Cy3 and Cy5 DIGE reagents. Figure supplied by GE Healthcare Fig. 4.3 Structures of the Cy3 and Cy5 DIGE reagents. Figure supplied by GE Healthcare however, this means that abundant proteins, such as albumin and immunoglobulins in mammalian plasma, are present and labelled with the expensive reagents. If fractionation of proteins is performed prior to 2-DE, such as depletion of abundant proteins, oxidative or photolytic damage to the fluorophors can occur in a...

Prefractionation Tools

The major limitations of available technologies for the complete investigation of any proteome have been highlighted in dozens of papers. Classical 2-DE techniques poorly represent strongly alkaline proteins, and they are not effective for the solubilisation, analysis and identification of highly hydrophobic proteins. However, since our first reviews of this area Herbert et al. 1997 , dramatic improvements in the analysis of both hydrophobic and alkaline proteins have been made. A greater...

Artefacts

As proteomics matures, there is a new focus on the analysis of co- and post-translational modifications. Modifications are one mechanism for the generation of complexity in the proteome, given the relatively small number of genes in the genomes of higher eukaryotes see Chap. 5 . Thus, separation scientists are under pressure to maximise resolution of modified proteins, while ensuring that artefactual protein modifications are eliminated or at least minimised during protein separation by 2-D...

Introduction

The broad workflow of proteomics usually encompasses protein extraction, separation and analysis by mass spectrometry. Resulting mass spectra are subsequently processed, and mass data are then matched with sequence data from databases to identify the proteins present. Thus, in a first approximation, the raw output from a proteomic study usually consists of a list of proteins along with accompanying expression profiles relevant to the experimental conditions under study. The characterisation of...

Multiplexed Approaches to Proteomics

Historically, proteome research has used three different approaches for the analysis of complex protein mixtures. First, and historically the original form of what is described as proteomics, is gel-based protein separation coupled with mass spectrometry analysis of the separated proteins. Second, and what has often been seen as a competing approach, is a range of liquid chromatography based protein or peptide separations and subsequent online electrospray mass spectrometry analysis of...