Observation of phosphorylation site-specific dissociation of singly protonated phosphopeptides

Abstract

In ultraviolet photodissociation of phosphopeptide ions with a basic residue (arginine, lysine, or histidine) at the N-terminus, intense a n Ϫ 97 peaks were observed. These ions were formed by cleavage at phosphorylated residues only. For multiply phosphorylated peptides, this site-specific cleavage occurred at every phosphorylated residue. H/D exchange studies showed that a n Ϫ 97 was formed by H 3 PO 4 loss from a n ϩ 1 radical cations. The site-specificity of phosphate loss observed here is in contrast to the nonspecific phosphate loss from b n and y n reported previously. Characteristics of the reaction and its potential utility for phosphopeptide analysis are discussed. (J Am Soc Mass Spectrom 2010, 21, 53-59)

Enhanced characterization of singly protonated phosphopeptide ions by femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fs-LID-MS/MS)

“…With the exception of the a 9 ϩ 1 ⌬ , x 7 ⌬ , and x 9 ⌬ product ions (where the superscript ⌬ corresponds to the loss of 98 Da, H 3 PO 4 ), all of the products formed via radical directed fragmentation pathways were observed to retain the phosphate moiety. As mentioned in the introduction, the observation of a n ϩ 1 ⌬ ions have recently been reported using 193 nm UVPD in a tandem time-of-flight (TOF-TOF) instrument, and can be used to provide characteristic information regarding the location of the phosphate group within the peptide sequence [27]. The formation of x n ⌬ ions identified in this study have not previously been reported.…”

“…In contrast, photodissociation in the ultraviolet (i.e., UVPD) wavelength range (157-355 nm; 7.9 -3.5 eV), by which prompt dissociation occurring from excited state species typically yields aor x-type product ions from singly-protonated peptides, as well as d-, v, and w-ions resulting from side-chain cleavages, depending on the location of the charge in the peptide and the observation time frame for the experiment [25,26]. Interestingly, it has been recently reported that 193 nm UVPD in a tandem time-of-flight (TOF-TOF) instrument yields characteristic a n ϩ 1-98 product ions when the n th residue is occupied by a phosphorylated species, suggesting that UVPD-MS/MS may offer unique diagnostic information for phosphorylation site assignment [27]. However, a recent study involving time resolved 157 nm UVPD of singly charged arginine-terminated phosphopeptides in an ion trap-TOF mass spectrometer revealed that abundant neutral losses of the phosphate group from the precursor ions are observed just 300 ns after photoactivation, and that phosphate group losses from the product ions became common after just 13 s [28].…”

Shedding Light on the Frontier of Photodissociation

“…All of the y ions C-terminal to the two crosslinked lysine residues were detected using IRMPD, and three b nβ product ions were detected, providing confirmation of the identity of the β peptide, L There have been several other recent reports of characteristic fragmentation pathways observed upon UVPD, typically catalyzed by the presence of specific functional groups or modified residues [20,28,29,31,37]. In one study, abundant a n -97 product ions were detected at each phosphorylated residue upon the UVPD (193 nm) of protonated phosphopeptides containing a basic amino acid at the N-terminus [20]. In another investigation, a specific C α -C β bond cleavage was reported for protonated tyrosinecontaining peptides upon UVPD at 262 nm, resulting in a radical product ion in which a specific portion of the tyrosyl residue was cleaved [37].…”

“…In contrast, the absorption of UV photons leads to the excitation of ions to higher electronic states and access to high-energy dissociation pathways not observed for CID or IRMPD. Photodissociation has been implemented in MALDI time-of-flight mass spectrometers for the analysis of singly charged ions [14][15][16][17][18][19][20][21][22][23] and ESI ion trapping instruments (both FTICR systems [24][25][26][27] and various quadrupole ion traps ) for the characterization of singly or multiply charged species. In general, time-of-flight instruments offer the advantages of high resolution, high accuracy, broad m/z range, and fast scan times, although photodissociation efficiencies in these mass spectrometers have been rather low due to the fact that irradiation of the ions occurs orthogonally in a very narrow time window as the selected ions exit the ion gating region.…”

“…A variety of lasers have been used for photodissociation, including CO 2 lasers (10.6 μm, 0.12 eV per photon) [24-27, 40-46, 48-52, 54-56, 61-63], F 2 excimer lasers (157 nm, 7.9 eV per photon) [14][15][16][17][18][19][20][21][22][23]39], ArF excimers (193 nm, 6.4 eV per photon) [57][58][59], Nd:YAG lasers (266 nm, 4.7 eV per photon [28][29][30][31][32], or 355 nm, 3.5 eV per photon [47,49]), femtosecond titanium sapphire lasers (800 nm, 1.5 eV per photon) [60], and OPO-Nd:YAG lasers that offer a tunable range from 205 nm to 2550 nm (6.0-0.49 eV per photon) [33][34][35][36][37]. As long as the laser provides sufficient power and offers a wavelength that will be absorbed by the analyte ions of interest, then it can be utilized for photodissociation.…”