Elsevier

Methods

Volume 35, Issue 3, March 2005, Pages 211-222
Methods

Mass spectrometry of peptides and proteins

https://doi.org/10.1016/j.ymeth.2004.08.013Get rights and content

Abstract

This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides in the gas phase after protein digestion, is discussed in more detail. The most widely used ion activation and dissociation process, gas-phase collision-activated dissociation (CAD), is discussed from a practical point of view. Infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) are introduced as two alternative dissociation methods. For spectral interpretation, the common fragment ion types in peptide fragmentation and their structures are introduced; the influence of instrumental methods on the fragmentation pathways and final spectra are discussed. A discussion is also provided on the complications in sample preparation for MS analysis. The final section of this article provides a brief review of recent research efforts on different algorithmic approaches being developed to improve protein identification searches.

Introduction

Mass spectrometry and tandem mass spectrometry (MS/MS) experiments are major tools used in protein identification. Mass spectrometers measure the mass/charge ratio of analytes; for protein studies, this can include intact proteins and protein complexes [1], fragment ions produced by gas-phase activation of protein ions (top-down sequencing) [2], [3], [4], [5], [6], peptides produced by enzymatic or chemical digestion of proteins (mass mapping) [7], [8], and fragment ions produced by gas-phase activation of mass-selected peptide ions (bottom-up sequencing) [9]. The application of mass spectrometry and MS/MS to proteomics takes advantage of the vast and growing array of genome and protein data stored in databases. The information produced by the mass spectrometer, lists of peak intensities and mass-to-charge (m/z) values, can be manipulated and compared with lists generated from “theoretical” digestion of a protein or “theoretical” fragmentation of a peptide. Applications to analyze ever smaller quantities of sample are driving the development of more sensitive mass spectrometers, as well as low flow, high resolution separation technologies, to provide structural information on individual components in complex mixtures of thousands of proteins derived from biological samples. Protein identification by mass spectrometry requires an interplay between mass spectrometry instrumentation (how molecules are ionized, activated, and detected) and gas-phase peptide chemistry (which bonds are broken, at what rate, and how cleavage depends on factors such as peptide/protein charge state, size, composition, and sequence). This brief tutorial article provides an overview of peptide and protein fragmentation in mass spectrometers.

Section snippets

Instrumentation

A rich variety of different MS/MS instrument configurations (with different capabilities in terms of speed, ionization method, resolution, sensitivity, and mass/charge range) have been developed both in research laboratories and in the marketplace for application to proteomics. (For a tutorial on mass spectrometry instrumentation, refer to http://staging.mc.vanderbilt.edu/msrc/tutorials/ms/ms.htm.) This large number of instrument types has developed because no one instrument type has all of the

Top-down sequencing: protein fragmentation in the gas phase

An approach that involves direct protein sequencing in the gas phase is referred to as top-down sequencing and has been demonstrated [2] and developed [3], [4], [5] over the past decade. This approach is an alternative to the commonly used bottom-up sequencing (see next section). In the top-down approach, the protein sample is not subjected to enzymatic digestion, but instead transferred into the gas phase intact. Subsequent measurement of the protein molecular weight and fragmentation of the

Bottom-up sequencing: peptide fragmentation in the gas phase

The more popular approach to protein identification relies on peptide sequencing and is referred to as bottom-up sequencing. This approach requires accurate sequence analysis of the MS/MS spectra of the proteolytic fragments so that protein identification can be made and typically relies on algorithms for amino acid sequence assignments.

Fragmentation mechanisms and algorithm development

In the early 1990s, computer search algorithms for identifying proteins from peptide mass spectral data became available allowing for the high-throughput identification of unknown proteins [68], [69], [70], [71], [72]. Pioneering studies in this area utilized in-gel digestion protocols and began to establish databases of proteins expressed in human myocardial cells, melanoma cells, and yeast [73], [74]. The term “proteomics,” used to describe the sum of proteins expressed in a given cell type,

Concluding remarks

In the last 15 years, mass spectrometry applications have revolutionized analysis of proteins, moving from simple studies of purified proteins, blocked N-termini, modified peptides, and analysis of peptide synthesis reactions, to the current dizzying array of new methods and instruments, as well as inspiration for the new field of systems biology. Proteomics is now a multibillion-dollar enterprise. In the same time, we have shifted from an era where our understanding of protein and peptide

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