No abstract

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve N™C ␣ cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population. ( ploys reactions of multiprotonated peptides/ proteins with low energy electrons to generate peptide fragments. The high sequence coverage and the ability to retain labile groups after ECD have allowed posttranslational modifications (PTMs) and point mutations (PMs) in peptides and proteins to be both identified and localized. Such performance is of great value for the analysis of the proteome, particularly that of diseased organisms. PTMs and PMs are widespread and are frequently associated with disease [5][6][7], for example over 80 different point mutations have been found in transthyretin (most of which lead to autosomal disorders) [8].To be able to fully characterize such modified proteins, 100% sequence coverage is required (so-called top-down proteomics). Several methods have been developed to increase the sequence coverage of ECD, which can be grouped into either infrared illumination [9 -11] or collisional activation [12]. These methods increase the average internal energy of the ions, which also affects the conformation of these gas-phase ions [13].There is experimental and theoretical evidence that ECD is directed by the internal solvation of the (neutralized) proton [9, 14 -18]. If the reaction progresses faster than the electron-proton recombination energy is randomized and thermal fluctuations are small, specific fragments would be expected from a single (frozen) conformer, reflecting the solvation shell, specific for that conformation, surrounding the neutralized proton. Thermal fluctuations that result in a dynamic solvation shell, but not conformational change (local fluctuations rather than a global change), would produce a greater number of fragments. Finally, multiple conformations as well as thermal fluctuations would produce yet more fragments. Because the methods used to increase the sequence coverage of ECD all increase the internal energy of the ions, and so affect the conformation of the gas-phase ions, the...

Tandem MS sequencing of peptides that contain a disulfide bond is often hampered when using a slow heating technique. We show that complexation of a transition-metal ion with a disulfide-bridge-containing nonapeptide yields very rich tandem mass spectra, including fragments that involve the cleavage of the disulfide bond up to 56% of the total product ion intensity. On the contrary, MS/MS of the corresponding protonated nonapeptides results predominantly in fragments from the region that is not involved in the disulfide bond. Eleven different combinations of three nonapeptides and three metal ions were measured using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) combined with sustained off-resonance irradiation collision induced dissociation (SORI-CID). All observed fragments are discussed with respect to four different types of product ions: neutral losses, b/y-fragmentation with and without the disulfide bond cleavage, and losses of internal amino acids without rupture of the disulfide bridge. Furthermore, it is shown that the observed complementary fragment pairs obtained from peptide-metal complexes can be used to determine the region of the binding site of the metal ion. This approach offers an efficient way to cleave disulfide-bridged structures using low energy MS/MS, which leads to increased sequence coverage and more confidence in peptide or protein assignments.

The application of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) for high resolution biomolecular analysis has increased greatly after 30 years of innovation since its conception in 1974. FT- ICR-MS can now routinely be used for the analysis of complex organic mixtures such as biological or petrochemical samples. Many of these new possibilities have been the results of many different instrumental developments. This paper provides a mini review of selected instrumental developments that now allow these measurements. The development of soft ionization techniques such as electrospray ionization and matrix assisted laser desorption and ionisation was crucial for the analysis of biological macromolecules. Improved ion transport optics led to an increase in sensitivity. New ICR cell designs complement the capabilities of FT-ICR-MS by allowing a more thorough study of the mechanism and kinetics of ion reactions in the gas-phase. A selected example of electron capture dissociation (ECD) employs these developments to investigate the role of peptide conformation in ECD. Improved electronics and software allow faster and more flexible experiments. All these improvements led to an increase in speed and sensitivity that are necessary to couple FT-MS to fast separation techniques such as nano-high performance liquid chromatography. The modern FT-ICR-MS instruments can be incorporated in virtual organizations allowing remote access to unique infrastructure. This concept of remote experimentation opens new possibilities for scientific collaborations between expert scientists at different locations and allows the efficient use of this expensive instrumentation.

A novel set-up for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is reported for simultaneous infrared multiphoton dissociation (IRMPD) and electron-capture dissociation (ECD). An unmodified electron gun ensures complete, on-axis overlap between the electron and the photon beams. The instrumentation, design and implementation of this novel approach are described. In this configuration the IR beam is directed into the ICR cell using a pneumatically actuated mirror inserted into the ion-optical path. Concept validation was made using different combinations of IRMPD and ECD irradiation events on two standard peptides. The ability to perform efficient IRMPD, ECD and especially simultaneous IRMPD and ECD using lower irradiation times is demonstrated. The increase in primary sequence coverage, with the combined IRMPD and ECD set-up, also increases the confidence in peptide and protein assignments. Proteomics is an indispensable technology in the molecular description of life's organization. As proteins carry out most biological activities in a cell or organism, 1 it is essential to characterize their structure, expression, distribution and interactions. Over the past decade mass spectrometry (MS) has become the method of choice for the systematic analysis of a proteome. It has enabled protein identification and quantification, protein profiling, and the characterization of protein interactions and modifications. Nevertheless, MSbased proteomics still faces significant challenges, 1,2 such as the improvement of tandem mass (MS/MS) spectra in order to decrease the amount of false positives in peptide and protein assignments. Here we describe an instrumental development to improve the quality and information content of MS/MS spectra. A common method of protein identification uses partial sequence information of one or more proteolytic peptides. 1,3,4 This sequence-tag strategy involves proteolysis of the protein mixture, followed by chromatographic separation of peptides and MS/MS. The peak lists from such MS/ MS spectra are then submitted to a database for protein identification.The confidence of the assignment increases with the amount of sequence information and the mass accuracy of the measurement. The high mass accuracy provided by Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) is the principal reason for it currently being the method of choice for proteomics investigations. 4 With suitable control, FTICRMS can provide mass accuracies consistently under 2 ppm, and the majority under 1 ppm. 5-7