Accurate Mass and PIRs: A New Strategy for Protein Interactions  James E. Bruce, Gordon A. Anderson, Nikola Tolic, Xiaoting Tang, Devi Adhikari, Gerhard Munske, Saiful Chowdhury  We have developed a novel approach for protein interaction profiling that involves a new type of chemical cross-linker. These compounds enable us to encode information at the MS/MS level to allow improved characterization of protein interactions in biological systems. Protein Interaction Reporters, or PIRs can be fragmented with high specificity and produce fragments with diagnostic masses. As a result, PIR ions increase our ability to identify cross-linked proteins with mass spectrometry, since the encoded mass provides information about the chemical functionality of the cross-linker. Furthermore, the specific cleavage of the PIR structure allows the release of intact peptide ions and subsequently, accurate peptide mass-based protein identification to be accomplished. This presentation highlights the PIR approach, the implementation in complex systems and data analysis strategies that are being developed to allow PIR optimization.

Protein-Protein Interaction Studies of Shewanella oneidensis MR-1  Devi P. Adhikari, Xiaoting Tang, Gerhard R. Munske, and James E. Bruce   Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium that can use a variety of soluble and insoluble substrates for deriving energy. This bacterium has the potential for bioremediation. However, it is not clear how this bacterium participates in novel electron transport mechanisms. Most cellular functions are carried out by multi-protein complexes. We developed a mass spectrometry identifiable novel PIR to study protein-protein interactions. Here, we report the preliminary results of protein-protein interaction studies on S. oneidensis cells using novel PIRs. We identified proteins using our PIR to create a protein database for the second stage. The second stage involves the study of the crosslinked peptides by LC/FTICR MS.

New Protein Interaction Reporters for Studying Protein-Protein Interactions  Saiful M. Chowdhury, Gerhard R. Munske, Xiaoting Tang, James E. Bruce   We have developed new cross-linkers to study protein-protein interactions. The first generation cross-linker of this kind that we call a Protein Interaction Reporter (PIR) was reported earlier this year (Tang et. al, Anal chem. 2005, 77, 311-318). The cross-linkers have been constructed with two reactive groups which are attached with two low energy MS/MS labile bonds. After fragmentation of the labile bonds, the middle part of the linker serves as a reporter ion to aid identification of cross linked peptides. We are investigating new features to improve the efficiency of release of reporter ions from cross-linked peptides. This presentation will highlight results obtained with several new compounds and their fragmentation characteristics.

Development of a Novel Ion Guide for Improved Ion Transmission in FTICR Mass Spectrometry Nathan K. Kaiser, Si Wu, Kai Zhang, David C. Prior, Michael A. Buschbach, Gordon A. Anderson, James E. Bruce One of the novel features in the development of our ICR mass spectrometer is the ion guide to transport ions from the ion accumulation region of the instrument to the ICR cell. Currently with conventional instruments, after the ions are trapped in the accumulation region, they are given kinetic energy and flown toward the ICR cell where they have to be re-trapped. This gives rise to time-of-flight issues were heavy ions fly slower than lighter ions, and all ions are not ejected from the ion accumulation region at the same time due to the spatial distribution of the ions and the lack of axial potential within the accumulation region. Also, these ions which were initially cooled in the accumulation region are given kinetic energy as they are flown toward the ICR cell. These higher kinetic energy ions form a less stable ion packet which leads to lower sensitivity and resolution in the mass spectra. The ions can be re-cooled by pulsing gas into the ICR cell. However, this leads to longer mass analysis time since there is a substantial delay between the pulse gas event and the excitation and detection events. The pulsed gas technique is ill-suited for highthroughput analyses. Our approach for ion transfer using segmented quadrupole rods (Restrained Ion Population Transfer or RIPT) gives us complete control of the ions from the time they enter the accumulation cell to the time they reach the ICR cell. This approach circumvents the problems of time-of-flight, and also minimizes the energy distribution to the ions, allowing us to transfer cooled ions from the ion accumulation region to the ICR cell.

A Two-Stage Mass Spectrometry Approach Using PIR Cross-Linkers for Global Protein-Protein Interaction Profiling  Xiaoting Tang, Devi Adhikari, Gerhard R. Munske, Kai Zhang, Nikola Tolic, Gordon A. Anderson, James E. Bruce  Mapping protein-protein interaction networks at systems level is essential for understanding molecular function in biological organisms. Cross-linking strategies for studying protein interactions are simple in principle; however, present tremendous challenges in reality due to inherent complexity from the reactions and the mass spectra. We aim to develop a novel strategy using special cross-linkers and a two-stage mass spectrometry approach for profiling protein-protein interactions on a global scale. We have reported previously a novel type of cross-linker, called Protein Interaction Reporter (PIR)1, that includes cleavable bonds with high specificities at low-energy MS/MS to allow release of a reporter tag and intact peptide chains. The second-generation PIR further incorporates a biotin tag for enrichment of cross-linked products. The two-stage mass spectrometry approach has been developed to capitalize on the advanced features of PIR cross-linkers and high performance of FT-ICR-MS instruments. Stage 1 focused on capturing cross-linked products at protein level and construction of a restricted database to be used for searching interacting proteins. Stage 2 involved enrichment of cross-linked peptides to be analyzed by multiplexed LC/FT-ICR-MS with low-energy CID activation on and off at alternating scans. To allow use of reporter ions as lock mass for internal calibration and PIR fragmentation pattern recognition that allows distinguishing inter-, intra-, or dead-end cross-linked peptides, ICR-2LS software and newly developed Excel-based algorithms (XLinks) were applied to analyze and search the data generated from stage 2. The cellular proteome of Shewanella oneidensis MR-1 was explored and investigated with this novel strategy.