Improved procedures for guanidination of lysine-containing peptides, a derivatization that results in increased MALDI mass spectral signal intensities are presented. The complete conversion of lysines to homoarginines can be accomplished in as little as 5 min. The method is demonstrated on a model peptide and on tryptic digests of three proteins. To demonstrate the applicability to proteomics samples, it is successfully applied to the digest of 50 fmol of a protein. Approaches for concentrating and purifying low-quantity protein digests following guanidination are evaluated. Experiments with the model peptide GRGDSPK enable investigation of the specificity of the guanidination reaction.

This article provides a perspective on collisions of ions with surfaces, including surfaceinduced dissociation (SID) and reactive ion scattering spectrometry (RISS). The content is organized into sections on surface-induced dissociation of small ions, surface characterization of organic thin films by collision of well-characterized ions into surfaces, the use of SID to probe peptide fragmentation, and the dissociation of large non-covalent complexes by SID. Examples are given from the literature with a focus on experiments from the authors' laboratory. The article is not a comprehensive review but is designed to provide the reader with an overview of the types of results possible by collisions of ions into surfaces. (J Am Soc Mass Spectrom 2008, 19, 190 -208) © 2008 American Society for Mass Spectrometry T andem mass spectrometry (MS/MS) is an essential tool for elucidating ion structure. The MS/MS experiment involves mass selection of a precursor ion followed by ion activation and subsequent dissociation. The ion activation step is commonly accomplished via collision-induced dissociation (CID) in which the initial kinetic energy of a projectile ion is converted into internal energy through inelastic collisions with a neutral gas. Several alternative activation methods have been used in tandem mass spectrometry, one of which is surfaceinduced dissociation (SID). SID is analogous to CID, except that a surface replaces the neutral gas as the collision target. A typical ion-surface collision event is illustrated in Figure 1.The incorporation of a surface into a mass spectrometer for ion activation was pioneered in the laboratory of R. Graham Cooks in the mid-1970s and early 1980s [1][2][3]. Since that time, collisions of lowenergy (eV) organic ions with surfaces within the tandem mass spectrometer have been valuable for analyzing surface composition, characterizing reactions between organic projectile ions and surface adsorbates, chemically modifying surfaces, and determining projectile ion structure. A major motivation for development of SID is that energy transfer to ionic projectiles can be improved by increasing the mass of the collision target. The total available energy for transfer into the internal modes of the projectile ion is defined by the center-of-mass energy (E COM ) described by eq 1:where E LAB is the laboratory collision energy and M ION and M N are the masses of the projectile ion and neutral, respectively. Energy transfer in CID is limited by the mass of the collision partner, typically inert gases such as helium, argon, or xenon. In SID, if one assumes that the surface is an infinitely large collision partner, E COM becomes independent of mass and approaches the laboratory collision energy. The assumption that the entire surface can be viewed as the collision partner is not always valid, however, and there are instances where the mass of the terminal groups on the surface influence the amount of energy transfer [4,5]. Nonetheless, the use of a massive surface target should, in theory, provide ...

In recent years mass spectrometry based techniques have emerged as structural biology tools for the characterization of macromolecular, non-covalent assemblies. Many of these efforts involve preservation of intact protein complexes within the mass spectrometer, providing molecular weight measurements that allow the determination of subunit stoichiometry and real-time monitoring of protein interactions. Attempts have been made to further elucidate subunit architecture through the dissociation of subunits from the intact complex by colliding it into inert gas atoms such as argon or xenon. Unfortunately, the amount of structural information that can be derived from such strategies is limited by the nearly ubiquitous ejection of a single, unfolded subunit. Here, we present results from the gas-phase dissociation of protein-protein complexes upon collision into a surface. Dissociation of a series of tetrameric and pentameric proteins demonstrate that alternative subunit fragments, not observed through multiple collisions with gas atoms, can be generated through surface collision. Evidence is presented for the retention of individual subunit structure, and in some cases, retention of non-covalent interactions between subunits and ligands. We attribute these differences to the rapid large energy input of ion-surface collisions, which leads to the dissociation of subunits prior to the unfolding of individual monomers.

Tryptic digests of three proteins are reacted with O‐methylisourea in order to convert lysine residues to homoarginines. The resulting homoarginine‐terminated peptides exhibit more intense MALDI mass spectral peaks than their lysine‐terminated predecessors. This simple chemical reaction should therefore facilitate protein sequencing and mass mapping. Copyright © 2000 John Wiley & Sons, Ltd.

Previous gas-phase dissociation experiments of protein-protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an "energy sudden" collision of protein-protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein-protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein-protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.