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RESEARCH PROFILES High power for mid-level proteomics How much resolution is enough? Bottom-up proteomics applications typically use lower-resolution instruments, such as ion traps, whereas top-down proteomics is often done on a high-resolution ion cyclotron resonance (ICR) instrument equipped with electron capture dissociation (ECD). In the May 15 issue of Analytical Chemistry (pp 3525–3534), Joshua Coon and co-workers at the University of Wisconsin Madison provide a glimpse of the “middle ground” with their description of modifications to a hybrid linear ion trap (LIT)–orbitrap mass spectrometer that accommodates electron transfer dissociation (ETD) for characterizing a broad range of large peptides and small proteins. The modifications are conceptually easy to implement and involve construction of a dual-emitter pulsed nanoelectrospray source, application of an rf trapping voltage to the LIT endcap electrodes, and addition of a power supply interfaced with the trap-control hardware. In positive-ion mode, the back end of the LIT is negatively biased, whereas the front and center sections are positively biased. The source, first described by McLuckey et al. in 2005, is pulsed to provide a spray of negative 9-anthracenecarboxylic acid ions. By segmenting the LIT’s voltages, negative ions in the front of the trap can be collisionally dissociated, generating decarboxylated anions capable of initiating ion–ion reactions; peptide cations remain trapped in the back end. Trap voltages are then equalized, and the two ion populations mix and undergo ETD before mass analysis. Detection was the tricky step, according to Coon. Products of the ETD reaction were either radially detected by the LIT electron multiplier or injected into the orbitrap on the back end of the LIT. “The z-type ions typically generated by ETD are fragile radical species,” explains Coon. “We didn’t know if they would survive injection into the orbitrap. That imparts a fair amount of energy to the ions, and we were worried they would
just fall apart.” Fortunately, the z 437.4655 ions held together. 5+ = 437.2658 Z 18 Z113+ = 437.9426 One drawback to the dual-spray 437.2652 437.6660 approach is that the %ETD, a 437.8662 measure of efficiency, is rather low—just 17% in one preliminary 437.4167 test. In contrast, a more “conventional” ETD approach with a stand-alone LIT and a negative437.0 438.0 436.5 437.5 438.5 439.0 439.5 chemical-ionization fluoranthene m/z source yielded more fragmenta3+ = 546.0147 Z 14 546.5800 tion, fewer proton transfer reac4+ 546.8309 Z 18 = 546.3304 546.3477 546.3291 tions, and a %ETD of 65%. In547.0819 546.6828 546.0133 creasing the amount of precursor 547.0175 547.3323 ions and/or the number of scans averaged helped offset the low ef546.6667 ficiency of the dual-spray-generated anions. Coon admits the decarboxylated 9-anthracenecarboxylic acid anions are a problem, because 546.0 546.5 547.0 547.5 m/z most of the signal they generate results in charge-state changes and Isobaric fragment ions are identified with the highnot in fragmentation. er-resolution orbitrap but are missed by standard The utility of the extended LIT analysis; thus, the two approaches yield signifimass range and high resolution of cant differences in sequence coverage and protein the orbitrap aided the analysis of identification scores. larger peptides, such as the septuply protonated ATCH peptide. lation times than the fluoranthene setup, Here, higher charge states and overlapping isotopic clusters were differentiated only 34 unique peptides were identified with the orbitrap, as compared with using the ~100,000 resolution. High 1031 peptides detected by conventional resolution and mass accuracy were also ETD. It’s a good start, and Coon bekey in identifying ubiquitin from its lieves the potential for the technique is dual-spray ETD-generated product ion spectrum. The identification expectation bright. “With better anions and a more sophisticated injection scheme, we can value from the orbitrap configuration –20 –7 was 10 , compared with 10 from the eliminate the long duty cycle.” It’s possible that ETD on the orbiconventional LIT setup; this reflected trap will compare favorably to ECD on the significant effect of high resolution an ICR—but at a much lower cost. “We in improving sequence coverage for need to do the head-to-head comparlarger, multiply charged fragments. isons,” Coon admits, “but the combina“One measure of the usefulness of a tion of ETD and orbitrap sensitivity will technique is whether or not you can do open a lot of doors. I think we will go it with chromatography,” Coon notes. from good performance to spectacular.” The researchers digested a yeast wholeThe orbitrap’s resolution may be too cell lysate with Lys-C, which generated a much for bottom-up proteomics and not complex mixture of large peptides, and quite enough for top-down proteomics, put the system through its paces. Bebut there is ample room in the middle cause the low efficiency of the decarfor this high-powered approach. a boxylated 9-anthracenecarboxylic acid anions required 20-fold-longer accumu—Karen R. Jonscher J U N E 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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