Proteomics

Tutorial on protein fingerprinting (Proteomics)

1. Introduction Mass spectrometry-based protein identification is one of the core techniques in proteomics. It is based on the comparison of peaks in mass spectra with calculated theoretical masses for proteins as derived from their primary structure. By cutting the protein to identify with a specific endoprotease or chemical reaction, a mixture of peptides is […]

Making nanocolumns and tips (Proteomics)

The successful production of nanocolumns and tips is vital to the success of an in-line multidimensional LC-MS/MS analysis. Making nanocolumns for LC/LC-MS/MS is a two-step process: first, fused-silica microcapillary tubing is heated and pulled to produce a column with a tip diameter of 2-5 |m, then the column is packed with chromatographic packing material. The […]

Separation-dependent approaches for protein expression profiling (Proteomics)

1. Introduction Intense efforts over the last few years have resulted in the availability, at the time of writing (February 2005), of complete genome sequences for 256 organisms (21 archael, 203 bacterial, 32 eukaryotic), including man. This wealth of information is an invaluable resource that will allow comprehensive studies of gene expression that will in […]

Separation-independent approaches for protein expression profiling (Proteomics)

1. Introduction The completion of the Human Genome Mapping Project plus many others has the potential to accelerate our understanding of the biology of these organisms. One of the most successful tools developed to exploit this new information is the cDNA or oligonu-cleotide microarray, capable of analyzing the relative level of transcription of thousands of […]

Two-dimensional gel electrophoresis (Proteomics)

1. Introduction Two-dimensional gel electrophoresis (2DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of new promising technologies that have emerged (MudPIT, stable isotope labeling, arrays) (see Article 13, Multidimensional liquid chromatography tandem mass spectrometry for biological discovery, Volume 5, Article 23, […]

ICAT and other labeling strategies for semiquantitative LC-based expression profiling (Proteomics)

1. Introduction Comprehensive proteome analysis requires the identification and quantification of all the proteins in an experimental or clinical sample. This is very much an ideal, because such samples often consist of thousands of proteins spanning a range of biophysical and biochemical properties, and that vary in abundance by many orders of magnitude. Fortunately, developments […]

Protein arrays (Proteomics)

1. Introduction High-density DNA microarray technology has played a key role in the analysis of whole genomes and their gene expression patterns. The ability to study many thousands of individual genes, using oligonucleotide or cDNA arrays, is now very widespread, with its uses ranging from the profiling of gene expression patterns in whole organisms or […]

2D DIGE (Proteomics)

1. Introduction Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) has been widely used over the past four decades to resolve several thousand proteins in a single sample. This has enabled the identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE has been used to compare relative […]

Detecting protein posttranslational modifications using small molecule probes and multiwavelength imaging devices (Proteomics)

1. Introduction The marriage of advanced imaging instrumentation and small-molecule detection reagents has been crucial to inventing new capabilities in electrophoretic detection, especially in the realm of nonradioactive posttranslational modification analysis. Fluorescent total-protein stains, such as SYPRO Ruby dye (Molecular Probes), Deep Purple dye (Amersham Life Sciences), and the cyanine dyes of the DIGE analysis […]

Image analysis (Proteomics)

1. Introduction Quantitative analysis of 2D gel electrophoresis is faced with a number of challenges. Although at first glance the resolution of 2-DE seems impressive, it is still not sufficient compared to the enormous diversity of cellular proteins. Comigrating proteins in the same spot are not uncommon (Figure 1a). In general, 2-DE has a maximum […]

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