Journal Title
Title of Journal: J Am Soc Mass Spectrom
|
Abbravation: Journal of The American Society for Mass Spectrometry
|
Publisher
Springer-Verlag
|
|
|
|
Authors: Shannon L Cook Glen P Jackson
Publish Date: 2011/01/29
Volume: 22, Issue: 2, Pages: 221-232
Abstract
The fragmentation behavior of nitrated and Snitrosylated peptides were studied using collision induced dissociation CID and metastable atomactivated dissociation mass spectrometry MADMS Various charge states such as 1+ 2+ 3+ 2– of modified and unmodified peptides were exposed to a beam of high kinetic energy helium He metastable atoms resulting in extensive backbone fragmentation with significant retention of the posttranslation modifications PTMs Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation ECD and electron transfer dissociation ETD MAD does produce numerous backbone cleavages in the vicinity of the modification Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues However compared with CID MAD produced between 66 and 86 more fragment ions which preserved the labile –NO modification MAD was also able to differentiate I/L residues in the modified peptides MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD ETD and CID for determination of PTMs such as nitrated and Snitrosylated peptidesElucidating how a PTM can alter a protein’s structure and biological function is an integral part of proteomics The ability to identify and localize these modifications in particular nitration and Snitrosylation can aid in biomarker discoveries for neurological diseases 1 2 cardiovascular diseases 3 4 lung diseases 5 6 and diabetes 7 Tyrosine and cysteine amino acid residues are highly susceptible to oxidation by one or more forms of reactive nitrogen species such as nitric oxide radicals 8 For example reaction of a nitrogen dioxide radical with tyrosine can produce a covalently bound –NO2 to the tyrosine side chain thereby producing 3nitrotyrosine 9 Cysteine nitrosylation can occur through a reaction with nitric oxide radicals resulting in the addition of –NO 10 Developing rapid and accurate mass spectrometry MSbased methods for direct detection of both nitration and Snitrosylation can aid in the understanding of how the reactive nitrogen species plays a role in certain diseases and can provide new diagnostic toolsMS has become a leading tool in proteomics because of its ability to sequence peptides and proteins and characterize PTMs The most widely used MS techniques for studying nitrotyrosine and nitrosylated cysteine PTMs in peptides and proteins are matrix assisted laser desorption/ionization timeofflight MS MALDITOF and electrospray ionization ESI MS/MS 9 UVMALDI has been noted to lead to a high degree of photodegradation or insource decay of nitrated or Snitrosylated peptides thereby rendering the modified peptides weak or absent 9 11 12 The dissociation method traditionally employed in ESI MS/MS is CID and CID tends to preferentially cleave side chain PTMs instead of the peptide backbone The preference for sidechain cleavages leads to poor sequencing information and PTM characterization Biotinswitching in which the labile nitrosylated cysteines are derivatized to a more stable biotinylated cysteine was developed to combat these issues 13 However the precise control of the chemical reactions has proven to be difficult and can result in false positives 12 14 Methods such as precursor ion scanning of the nitrotyrosine immonium ions 15 and selected reaction monitoring combined with UV absorption 16 have both shown to be promising techniques to characterize nitrotyrosine when analyzing complex samplesAlternative types of peptide dissociation methods that utilize radical chemistry have been recently introduced to provide both accurate sequencing and PTM information ECD first introduced in 1998 by McLafferty and coworkers has proven to effectively fragment the peptide backbone in Fourier transform ion cyclotron resonance mass spectrometers FTICRMS 17 This method preferentially cleaves the more energetic peptide backbone N–Cα bonds resulting in mainly c/ztype ions in contrast to the “slowheating” dissociation methods such as CID and IRMPD 18 The latter activation methods tend to cleave the lower energy C–N bonds and produce b/ytype ions ETD introduced by Hunt and coworkers uses ion–ion reactions to transfer electrons from a radical anion to polycations resulting in ECD like fragmentation 19 Successful PTM site determinations of phosphorylation 19 20 sulfation 21 22 and glycosylation 23 24 25 among others have been reported for ECD and ETD However traditional ECD and ETD are not applicable to singly protonated or negatively charged precursor ions such as the more acidic peptides Recently new techniques have been developed to apply these two methods NETD and niECD to negative ions 26 27 28There have been only a few reports applying ECD and ETD to nitrated and Snitrosylated peptides It has been shown that in ECD and ETD backbone fragmentation is completely inhibited in peptides containing specific tags with electron affinities EA larger than 1 eV Such tags are termed electron predators and examples include 3nitrobenzyl and 35dinitrobenzyl moieties 29 Beauchamp and coworkers proposed that the propagation of the captured electron can relax through space or through bond π to high EA predators tags which competes with the traditional electron transfer to the amide π orbital A hydrogen atom from a separate site on the peptide can then be transferred to the electron predator radicals due to their high Hatom affinity Tureček on the other hand proposes intermolecular Hatom transfer from the aminoketyl radical of the chargereduced species to the nitrobenzyl moieties instead of direct electron sequestering 30 In this mechanism backbone fragmentation is inhibited based on the high Hatom affinity of the nitrobenzyl groups Regardless of the exact mechanism the presence of nitrobenzyl moieties inhibits traditional ECD and ETD N–Cα backbone cleavage Recently nitrotyrosine residues have been shown to hinder backbone cleavage during ECD because of the electron predator or Hatom trap effect 31 These results demonstrate that ECD is not the method of choice to characterize nitrated tyrosine residues of 2+ and 3+ charge states of peptides 31 However topdown analysis of multiply charged larger nitrotyrosinecontaining proteins using ECD has shown promise when combined with other “slow heating” methods to identify PTM sites 32Few studies have concentrated on direct MS detection of cysteine nitrosylation probably due to its labile nature Snitrosylated bovine insulin was explored with ETD resulting in only a select number of backbone cleavages and fragment ions retaining the modification thereby hindering site determination 22 The major dissociation pathway observed in ECD was charge reduction electron transfer without fragmentation with and without losses of the NO 22 ETD was therefore unable to identify the location of the PTMRecently a new type of dissociation method using metastable atoms as the electron vehicle or potential energy source has been explored in RF ion trapping instruments which we term metastable atomactivated dissociation MAD The interaction of isolated precursor ions with a high kinetic energy beam of argon or helium metastable atoms produces a high degree of peptide backbone cleavages resulting in a b c x y and ztype ions while retaining PTMs 33 To date comprehensive dissociation has been demonstrated to fragment multiply charged cations and anions 33 34 35 1+ cations 33 36 37 phosphorylated cations 33 35 37 disulfide bonds 35 to cleave the amide ring structure of proline 33 and to differentiate isoleucine from leucine 33 36
Keywords:
.
|
Other Papers In This Journal:
|