Defining the Disulfide Bonds of Insulin-like Growth Factor-binding Protein-5 by Tandem Mass Spectrometry with Electron Transfer Dissociation and Collision-induced Dissociation

Abstract

Background: Only 2 of 9 putative disulfide bonds have been mapped for IGFBP-5. Results: Using a MS-based strategy combining ETD and CID, and ab initio molecular modeling, we have mapped all 9 disulfide bonds in IGFBP-5. Conclusion: Our results provide new insights into the IGFBP-5 structure. Significance: We define an approach using tandem MS and ab initio molecular modeling to characterize unknown disulfide linkages in proteins.

Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex

“…Analysis of iodoacetic acidtreated, non-reduced proteins showed no alkylation of any Cys-containing peptides, strongly indicating that no free Cys-SH was present (data not shown). Furthermore, tryptic peptide mass fingerprinting of non-reduced protein complexes identified Cys-Cys crosslinked peptides consistent with the previously reported IGFBP disulfide structures for the N-terminal domain of IGFBP4 and IGFBP5 and the C-terminal domain of IGFBP2 (and other IGFBPs; Smith et al 1989, Kalus et al 1998, Zesławski et al 2001, Mark et al 2005, Siwanowicz et al 2005, Sitar et al 2006, Brown et al 2008, Nili et al 2012. This pattern can be described by numbering the Cys residues consecutively.…”

Proteomic analysis and molecular modelling characterize the iron-regulatory protein haemojuvelin/repulsive guidance molecule c

“…Moreover, with the assistance of unconstrained ab initio molecular modeling we assigned the other 4-disulfide bonds to a cysteine-rich region of the protein [31]. Here we have applied an analogous experimental plan to HJV/RGMc, a protein for which no structural information is available.…”

Selective cleavage upon ETD of peptides containing disulfide or nitrogen–nitrogen bonds

“…The prominence of disulfide bond cleavage was first noted upon ECD in the late 1990's [12], and the preferential cleavage was attributed to the high hydrogen atom affinity of the SÀ ÀS functionality. This propensity for SÀ ÀS bond cleavage was also noted upon ETD [10], and since then a number of other groups have reported the same phenomenon upon ECD or ETD for both multi-protonated peptides and proteins and even upon CID [16][17][18][19][20][21][22][23][24][25][26], infrared multiphoton dissociation (IRMPD) and electron detachment dissociation (EDD) of deprotonated peptides that contain disulfide bonds [9]. The mechanistic details of SÀ ÀS bond cleavage upon electron activation of peptides have been explored in some detail, and it was determined that SÀ ÀS bonds were particularly prone to electronattachment cleavage because of the relatively low energies of the SÀ ÀS s * anti-bonding orbitals [27].…”

“…Cleavage of specific bonds offers significant potential for targeted proteomic applications, such as for identification of key post-translational modifications or for profiling selected classes of peptides based on cleavable tags. Despite this strategic potential, only a handful of studies have reported preferential bond cleavages [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Bond-selective cleavages are uncommon in part because of the fast re-distribution of internal energy that occurs for the more prevalent collisionbased activation methods which eliminates the likelihood of localized energy accumulation in specific bonds and in part because of the low frequency of highly labile bonds in peptides.…”

“…One of the most notable examples of preferential bond cleavage upon MS/MS is disulfide cleavage which has been reported upon ECD [12], ETD [10], UVPD at 157 nm [14], UVPD at 266 nm [15], and even upon CID to a lesser extent [16][17][18][19][20][21][22][23][24][25][26]. The prominence of disulfide bond cleavage was first noted upon ECD in the late 1990's [12], and the preferential cleavage was attributed to the high hydrogen atom affinity of the SÀ ÀS functionality.…”