Overview of Peptide and Protein Analysis by Mass Spectrometry

Abstract

The goals of this unit are to familiarize peptide and protein chemists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. The emphasis here is on established tools and techniques that can realistically be used for problem solving. As a result, many useful instruments and techniques employed by the trained mass spectroscopist are not discussed here, because they do not satisfy these criteria or are not yet widely available. A few of these are briefly mentioned at the end of this introductory overview. The overview concludes with a tutorial discussion on the meaning and practical importance of a number of fundamental experimental and performance-related issues that have important implications for interpretation and use of mass spectral data, such as mass accuracy and resolution.

Development of a Novel Proteomic Approach for the Detection of Transitional Cell Carcinoma of the Bladder in Urine

“…confirmation of quantitative differences by mass spectrometry is not yet standardized and, although feasible, is technically very challenging. 22 The development of a reliable method of protein quantification as well as the application of different types of chip chemistries that promises to increase the resolving power of the assay, are ongoing efforts to detect additional normal-as well as TCC-associated urinary proteins. Searching the protein databases suggested that the 3.3/3.4-kd TCC-associated protein (biomarker UBC1) might be a member of the defensin family of peptides.…”

“…[21][22][23] Although the resolving power of 2D gels remains unchallenged, the high sensitivity, speed, and reproducibility of mass spectrometry have boosted its application in all aspects of protein analysis, including discovery, identification (ie, peptide mapping, sequencing), and structural characterization. Analogous to the DNA chip technologies that allow the study of gene expression profiles, Ciphergen Biosystems, Inc. (Fremont, CA) has recently developed the ProteinChip technology coupled with SELDI-TOF-MS (surface-enhanced laser desorption/ionization time of flight mass spectrometry) to facilitate protein profiling of complex biological mixtures.…”

Mass Spectrometry in Proteomics

“…MS, in particular the application of ESI coupled on-line with high-performance separation techniques such as capillary electrophoresis (CE) and HPLC, has had a dramatic effect on the sensitivity and the speed with which the primary structure of proteins and peptides can be determined. Advances in separation techniques, particularly their implementation in miniaturized formats on-line with high-performance mass spectrometers, − and the development of miniaturized sprayers as ESI ion sources − have reduced the amount of peptide required for complete and routine sequence characterization from several picomoles of peptide , in the mid-1980s to a few femtomoles and below by the mid-1990s. − The development of mass spectrometric techniques of yet higher throughput and sensitivity is an essential component of the emerging field of proteomics and is still forcefully pursued today. Through incremental improvements in on-line separation methods, sensitivities in the sub-femtomole peptide detection and identification range have been achieved with commercially available ion trap mass spectrometers. , For simple mass measurement, sensitivities into the zeptomole (10 -21 mol) range have been observed with prototype FT-ICR-MS instruments .…”

“…Advances in separation techniques, particularly their implementation in miniaturized formats on-line with high-performance mass spectrometers, [45][46][47][48][49][50] and the development of miniaturized sprayers as ESI ion sources [51][52][53] have reduced the amount of peptide required for complete and routine sequence characterization from several picomoles of peptide 54,55 in the mid-1980s to a few femtomoles and below by the mid-1990s. [56][57][58][59] The development of mass spectrometric techniques of yet higher throughput and sensitivity is an essential component of the emerging field of proteomics and is still forcefully pursued today. Through incremental improvements in on-line separation methods, sensitivities in the sub-femtomole peptide detection and identification range have been achieved with commercially available ion trap mass spectrometers.…”

Proteomic analyses of arsenic‐induced cell transformation with SELDI‐TOF ProteinChip® technology

“…Significant technological advances in protein chemistry in the last decade have established mass spectrometry as an indispensable tool for protein study [Carr et al, 1991;Carr and Annan, 1998;Patterson, 1998]. Although the resolving power of 2D gels remains unchallenged, the high sensitivity, speed, and reproducibility of mass spectrometry have boosted its application in all aspects of protein analysis, including discovery, identification (i.e., peptide mapping, sequencing), and structural characterization.…”