Protein identification via surface-induced dissociation in an FT-ICR mass spectrometer and a patchwork sequencing approach

Abstract

Surface-induced dissociation (SID) and collision-induced dissociation (CID) are ion activation techniques based on energetic collisions with a surface or gas molecule, respectively. One noticeable difference between CID and SID is that SID does not require a collision gas for ion activation and is, therefore, directly compatible with the high vacuum requirement of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Eliminating the introduction of collision gas into the ICR cell for collisional activation dramatically shortens the acquisition time for MS/MS experiments, suggesting that SID could be utilized for high-throughput MS/MS studies in FT-ICR MS. We demonstrate for the first time the utility of SID combined with FT-ICR MS for protein identification. Tryptic digests of standard proteins were analyzed using a hybrid 6-tesla FT-ICR mass spectrometer with SID and CID capabilities. SID spectra of mass-selected singly and doubly charged peptides were obtained using a diamond-coated target mounted at the rear trapping plate of the ICR cell. The broad internal energy distribution deposited into the precursor ion following collision with the diamond surface allowed a variety of fragmentation channels to be accessed by SID. Composition and sequence qualifiers produced by SID of tryptic peptides were used to improve the statistical significance of database searches. Protein identification MASCOT scores obtained using SID were comparable or better than scores obtained using sustained off-resonance irradiation collision-induced dissociation ( A fter the success of the genomics projects, proteomics has become a major research enterprise with the objective to study the functionality and dynamics of protein expression, the correlation between complementary genomes and proteomes, and posttranslational modifications of proteins that cannot be studied by genomics alone [1]. A major focus in proteomics is protein identification, a task that has been significantly accelerated by the advent of modern mass spectrometric technologies [2].Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the highest mass resolution and mass accuracy of present MS technologies and, despite its large size and relatively high purchase and maintenance cost, is now considered one of the most powerful techniques in proteomics research [3]. In the majority of FT-ICR MS proteomic applications, peptide ion activation is achieved by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) [4]. SORI-CID results in sequential activation of the precursor ions by multiple low kinetic energy collisions with a collision gas that is pulsed into the ICR cell. The fast rise in pressure produced by the injection of this collision gas is followed by a long pump down delay (typically 2 to 5 s) to enable fragment ion mass analysis, significantly slowing down the acquisition of MS/MS spectra.In proteomic applications requiring the separation of complex peptide mixtures, FT-ICR MS is commonly coupled ...

Surface-induced dissociation of small molecules, peptides, and non-covalent protein complexes

“…These qualifiers, consisting mainly of a 2 , b 2 , y 1 , y 2 , and internal and immonium type ions, provide additional restraints during database searching that help ensure the correct peptide is identified. Fernandez et al [87] demonstrated that SID in an FT-ICR yielded a large number of these qualifier ions, improving the statistical significance of database searches. In addition, SID conducted on a diamond surface provided improved sequence coverage over SORI-CID resulting from the accessibility of both fast and slow dissociation pathways not readily accessible by CID.…”

Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure

“…7,15 The use of these easy-to-prepare surfaces has persisted, although several other surface types have been utilized. 16–24 These SAMs provide a large effective mass for collision of projectile ions, the fluorocarbon chains are relatively rigid so that they don’t severely dampen the energy of the colliding projectile, and the fluorocarbon resists facile electron transfer to the incoming ions. Surface targets, in contrast to the typical gaseous targets such as Ar that are used for the more common activation method collision-induced dissociation (CID), have proven to be exceptionally useful for the characterization of protein complex quaternary structure.…”

Uncovering the Stoichiometry of Pyrococcus furiosus RNase P, a Multi‐Subunit Catalytic Ribonucleoprotein Complex, by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

“…Because Mg 2+ can stabilize protein and RNP complexes [17] and create structures that are difficult for CID to break apart, SID was used to dissociate RNPs in the presence of Mg 2+ . SID deposits high energy into the ions through a single collision event with a surface [18] to reveal dissociation pathways with high energy barriers and high rates, which are likely for RNP disassembly. In tandem with IM, SID can be employed to dissociate a putative RNP complex, thereby confirming its identity and revealing substructures.…”