We discuss the evolution of Orbitrap mass spectrometry (MS) from its birth in the late 1990s to its current role as one of the most prominent techniques for MS. The Orbitrap mass analyzer is the first high-performance mass analyzer that employs trapping of ions in electrostatic fields. Tight integration with the ion injection process enables the high-resolution, mass accuracy, and sensitivity that have become essential for addressing analytical needs in numerous areas of research, as well as in routine analysis. We examine three major families of instruments (related to the LTQ Orbitrap, Q Exactive, and Orbitrap Fusion mass spectrometers) in the context of their historical development over the past ten eventful years. We discuss as well future trends and perspectives of Orbitrap MS. We illustrate the compelling potential of Orbitrap-based mass spectrometers as (ultra) high-resolution platforms, not only for high-end proteomic applications, but also for routine targeted analysis.

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.

Modern ion trap mass spectrometers are capable of collecting up to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition speeds that can sample every eluting peptide precursor presented to the MS system. In practice, however, the precursor sampling capacity enabled by these ultrafast acquisition rates is often underutilized due to a host of reasons (e.g., long injection times and wide analyzer mass ranges). One often overlooked reason for this underutilization is that the instrument exhausts all the peptide features it identifies as suitable for MS/MS fragmentation. Highly abundant features can prevent annotation of lower abundance precursor ions that occupy similar mass-to-charge (m/z) space, which ultimately inhibits the acquisition of an MS/MS event. Here, we present an advanced peak determination (APD) algorithm that uses an iterative approach to annotate densely populated m/z regions to increase the number of peptides sampled during data-dependent LC-MS/MS analyses. The APD algorithm enables nearly full utilization of the sampling capacity of a quadrupole-Orbitrap-linear ion trap MS system, which yields up to a 40% increase in unique peptide identifications from whole cell HeLa lysates (approximately 53 000 in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved peptide and protein identifications across several modes of proteomic data acquisition, including varying gradient lengths, different degrees of prefractionation, peptides derived from multiple proteases, and phosphoproteomic analyses. Additionally, the use of APD increases the number of peptides characterized per protein, providing improved protein quantification. In all, the APD algorithm increases the number of detectable peptide features, which maximizes utilization of the high MS/MS capacities and significantly improves sampling depth and identifications in proteomic experiments.

Intact protein sequencing by tandem mass spectrometry (MS/MS), known as top-down protein sequencing, relies on efficient gas-phase fragmentation at multiple experimental conditions to achieve extensive amino acid sequence coverage. We developed the "topdownr" R-package for automated construction of multi-modal (i.e. involving CID, HCD, ETD, ETciD, EThcD and UVPD) MS/MS fragmentation methods on an orbitrap instrument platform and systematic analysis of the resultant spectra. We used topdownr to generate and analyze thousands of MS/MS spectra for five intact proteins of 10-30kDa. We achieved 90-100% coverage for the proteins tested and derived guiding principles for efficient sequencing of intact proteins. The data analysis workflow and statistical models of topdownr software and multi-modal MS/MS experiments provide a framework for optimizing MS/MS sequencing for any intact protein. Refined topdownr software will be suited for comprehensive characterization of protein pharmaceuticals and eventually also for de novo sequencing and detailed characterization of intact proteins.

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications. Molecular & Cellular Proteomics 9:824 -837, 2010.Over the last two decades, the integration of the unanticipated discovery of new modes of internal energy deposition and rapid technological progress in mass spectrometrybased instrument platforms has revolutionized our experimental capabilities to effectively study the inherent complexity of human and other mammalian proteomes. This includes the rapid identification of proteins, the detection and unambiguous assignment of protein isoforms, and the detection and localization of post-translational modifications (PTMs). 1The vast majority of mass spectrometry-based studies in protein biology and proteomics has used proteolytic digestion using trypsin and analysis of the resulting peptide mixtures by a variety of MS and MS/MS approaches (1-4). Although of considerable use for many purposes, this common strategy has a variety of limitations especially from the view of acquiring the information needed to establish and understand biological function. These limitations include the loss of information regarding the nature of protein isoforms and a loss of knowledge of the global presence of multiple PTMs that can co-occur at differing sites on the same protein molecule. These deficiencies are exacerbated when the portions of protein sequence coverage observed in a digest are low because peptides that would reveal the modifications or isoform-specific sequence variances may not have been detected. Additionally, the sequence identification for fragment ion spectra representing peptides with modifications that are unanticipated can be difficult to assign using common search algorithms.Hence, structural information covering an entire protein sequence has been lacking, and although needed from a protein biology viewpoint all along, the kind of methodology that would be required to obtain this knowledge has begun to emerge only recently. This has come about through the discovery of electron capture by polyprotonated peptide and protein species (5) and the more recent development of electron transfer by their reaction with suitable radical anions (6). Because these physicochemical processes deposit sufficient internal energy at the sites of charge reduction to cleave peptid...