Data Availability StatementData availability: Supplementary data is available with this publication at the MCP web site. power to 240,000 at 400 for a 768 ms transient. For top-down experiments, we combine GSK343 price a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 sincreasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides. In many mass spectrometric applications, the resolving power of the instrument is of pivotal importance. Ultimate resolution has so far been obtained by Fourier Transform Mass Spectrometry (1) Rabbit Polyclonal to SLC25A6 and in a recent example, Marshall and co-workers detected more than 26,000 components in a single spectrum of a crude oil mixture (2). In ion cyclotron resonance (ICR)1 Fourier transform mass spectrometry, resolution is determined by the length of the transient and by the strength of the magnetic field. Increasingly larger magnets have allowed resolution in excess of one million for small molecules. The relatively recently introduced OrbitrapTM analyzer utilizes a different physical principle to obtain high resolution (3C6). The signal is recorded from the image current produced by ion packets which oscillate around and along the spindle-shaped inner electrode of the trap: the higher the electric field, the larger the number of oscillations per unit time and the higher the resolving power. To increase field strength, several design options can be pursued, including increasing the radius of the inner electrode of the device (7). Here we describe an Orbitrap analyzer that achieves higher resolving power through reduced trap dimensions. Resolution is further increased by making use of the phase information during Fourier Transformation (8C11). This ultra high GSK343 price resolution Orbitrap analyzer was combined with other instrumental improvements to construct a novel linear ion trap Orbitrap hybrid mass spectrometer termed the Orbitrap Elite. We describe principles of this instrument and characterize its operation for both intact protein analysis and for bottom up GSK343 price peptide mixture analysis. Top down protein analysis has previously mainly been performed with Fourier transform (FT) ICR instruments because of their very high resolving power (12C14). One of the challenges in using top down approaches in proteomics has been to obtain cycle times commensurate with liquid chromatography tandem MS (LC MS/MS) time scales (15). The linear ion trap Orbitrap has also been employed for top down proteomics (16C19). Here we take advantage of the ultra high resolution of the Orbitrap Elite to enable fast LC MS/MS compatible top-down scan methods. In bottom-up proteomics typically very complex peptide GSK343 price mixtures are analyzed (20C22). Online LC MS runs contain evidence for tens of thousands of peptides (23, 24) and this places a premium on the resolution of the survey (MS) scans. A popular shotgun proteomics method on the linear ion trap Orbitrap (LTQ Orbitrap or LTQ Orbitrap Velos) is a 1 s survey scan with 60,000 resolution at 400 (768 ms transient), and ion trap collision-induced dissociation (CID) scans of the ten or twenty most abundant ions (high resolution low resolution or highClow top10 method). Here we explore topN methods with much higher resolution survey scans as well as an increased number of fragmentation events per cycle enabled by rapid CID scans. A highChigh strategy (high resolution MS as well as MS/MS (25)) has been routinely made possible on Orbitrap instruments by higher energy collisional dissociation (HCD) with the advent of the LTQ-Orbitrap Velos (26). We show that this strategy benefits from the shorter transients and higher resolving power possible on the Orbitrap Elite. It has been demonstrated that a combination of two fragmentation methods can greatly augment sequence related information in peptide MS/MS (27C29) and we explore this dual approach with CID and HCD fragmentation of the same precursor ions. EXPERIMENTAL PROCEDURES The Orbitrap Elite is a further.