Ultra High Resolution Linear Ion Trap Orbitrap Mass Spectrometer (Orbitrap Elite) Facilitates Top Down LC MS/MS and Versatile Peptide Fragmentation Modes

Abstract

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm—incorporating phase information—further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine 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 s—increasing 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.

Comprehensive Identification of Proteins from MALDI Imaging

“…To the best of our knowledge, it is the first time that this has been successfully demonstrated on MALDI IMS samples. The top-down analysis has been greatly aided by the short list of proteins identified from the tissues and assembled in the subsetDB and by employing a very high performance mass spectrometer (36).This subsetDB is 4ϫ smaller than Swissprot and 15ϫ smaller than the original human IPI database commonly used in proteomics (37) and thus vastly reduces the number of proteins that have to be considered in a database search and increases the confidence in peptide identification scores. Because no protease can be specified in the search, the very high mass accuracy afforded by the mass spectrometer (single digit ppm) for intact peptides as well as fragment ions together with the small sequence database enables searching the data in a reasonable time and reduces the number of possible false positive identifications (38).…”

“…Progress in the performance of mass spectrometers is now making this more readily possible (36) and the short-list of MALDI IMS proteins generated in this study is of tremendous help for this purpose. As a proof-of-concept, we prepared MALDI matrix extracts from esophagus and colon tissue sections, mixed the extracts and analyzed these by LC-MS/MS on a high field Orbitrap mass spectrometer (36). Tandem mass spectra were charge deconvoluted and deisotoped before searching against a collection of ϳ5,500 protein sequences assembled from protein identifications made in MALDI IMS studies including the ones presented above (subsetDB, supplemental Files S1 and S2).…”

Mesenchymal Stem Cell Therapy Protects Lungs from Radiation-Induced Endothelial Cell Loss by Restoring Superoxide Dismutase 1 Expression

“…Experiments were performed on an Orbitrap Elite instrument (Thermo Fisher Scientific, Waltham, MA) that was coupled to an EASY-nLC 1000 liquid chromatography (LC) system (56). The LC was operated in the two-column mode.…”

Top-down proteomics: applications, recent developments and perspectives

“…Michalski et al reported a compact high-field orbitrap, coupled with an improved Velos PRO dual ion trap mass spectrometer and advanced signal processing, capable of a nearly 4-fold increase in resolution [87]. Top-down proteomic analysis of H1299 human cancer cell line proteome using this instrument has also been reported and identified 690 unique proteins from the H1299 human cancer cell line [88].…”