Modern Mass Spectrometry in Protein Analysis - - Chromatography Online
Modern Mass Spectrometry in Protein Analysis

The Column
Volume 10, Issue 15, pp. 18–19

An excerpt from LCGC's e-learning tutorial on MS in protein analysis at http://CHROMacademy.com/


Photo Credit: LAGUNA DESIGN/Getty Images
It has been 25 years since electrospray ionization was shown to lead to multiple charging of peptides and proteins, effectively bringing these large biomolecules into the measurable range of the common mass analyzing devices.

In modern times, mass spectrometry (MS) is widely used in the characterization of proteins at the intact (native) and peptide levels, as well as in the analysis of enzymatically generated protein fragments, in conjunction with an expanding range of compatible chromatographic separation techniques.

Electrospray ionization of intact proteins generates a charged envelope of peaks corresponding to differing degrees of ionization that can readily be converted to the molecular mass using a mathematical algorithm. Typically, average mass is calculated; however, high mass accuracy time-of-flight and Fourier transform instruments allow isotopic resolution, which can be important in the study of protein fragments to determine sequence and post-translational modifications (PTMs). This has become known as "top-down" proteomics, because the measurements are made on the intact proteins instead of enzymatically digested proteins. These methods require special ionization techniques such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD), which typically maintain the labile modifications (glycosolation for example) on the protein backbone.


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