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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Mass Spectrometry Behavior of Modified Peptides

MS Fragmentation Modes

Fragmentation of peptides detected in the MS survey scan (MS1) to generate product ion spectra plays an important role in the identification and localization of PTM. The key requirement is to generate sufficient fragment ion information to accurately assign acetylation and/or methylation status and to determine the amino acid sequence of the precursor for site assignment.

Collision-induced dissociation (CID) and increasingly higher-energy colli- sional activation/C-trap dissociation (HCD) are both used in the large majority of proteomics experiments for peptide identification following proteolytic digestion. CID and HCD fragmentations occur along the peptide backbone at the amide bonds to generate b and y ions, which contain the N- and C-termini, respectively. Amino-immonium (IM) ions, which are unique for the majority of naturally occurring amino acids, except lysine/glutamine or leucine/isoleu- cine, confirm the presence of specific amino acids. In general, CID and HCD are most effective for low-charged tryptic peptides (typically 9-12 amino acids), which are doubly and triply charged. Trypsin cleavage is highly specific and reproducible, cleaving C-terminal to lysine and arginine residues, except when lysine and arginine have proline C-terminal to their position or are N-linked to aspartic acid. Typically, CID or HCD are used either alone or in combination with electron transfer dissociation (ETD) for PTM-directed analytical workflows [22]. ETD provides an alternative fragmentation strategy to CID and generates complementary sequence information to y- and b-type ions resulting from CID and HCD [26]. ETD cleavage occurs at the Ca-N bond producing c- and z-type ions, with a higher tendency for retention of PTM side-chain groups relative to CID/HCD, a particular benefit to PTM site localization [27]. ETD also has compatibility with higher charge state peptides such as those characteristic of both miscleaved tryptic peptides (which can result from acetylation or methylation) and nontryptic peptides, for example, Arg-C, Glu-C, Asp-N, and chymotrypsin-generated peptides [26]. The use of alternating CID/ETD has been shown to be beneficial for the detection of acetylation- and methylation-modified peptides [28, 29]. The fragmentation of longer peptides and proteins by ETD has also found application for top- down and middle-down proteomics studies (see Section 4.3).

 
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