Targeted mass spectrometry methods produce high-quality quantitative data in terms of limits of detection and dynamic range, at the cost of a substantial compromise in throughput compared to methods ...such as data independent and data dependent acquisition. The logistical and experimental issues inherent to maintaining assays of even several hundred targets are significant. Prominent among these issues is the drift in analyte retention time as liquid chromatography (LC) columns wear, forcing targeted scheduling windows to be much larger than LC peak widths. If these problems could be solved, proteomics assays would be capable of targeting thousands of peptides in an hour-long experiment, enabling large cohort studies to be performed without sacrificing sensitivity and specificity. We describe a solution in the form of a new method for real-time chromatographic alignment and demonstrate its application to a 56 min LC-gradient HeLa digest assay with 1489 targets. The method is based on the periodic acquisition of untargeted survey scans in a reference experiment and alignment to those scans during subsequent experiments. We describe how the method enables narrower scheduled retention time windows to be used. The narrower scheduling windows enables more targets to be included in the assay or proportionally more time to be allocated to each target, improving the sensitivity. Finally, we point out how the procedure could be improved and how much additional target multiplexing could be gained in the future.
Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' ...(lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.
Parallel reaction monitoring (PRM) is an increasingly popular alternative to selected reaction monitoring (SRM) for targeted proteomics. PRM’s strengths over SRM are that it monitors all product ions ...in a single spectrum, thus eliminating the need to select interference-free product ions prior to data acquisition, and that it is most frequently performed on high-resolution instruments, such as quadrupole-orbitrap and quadrupole-time-of-flight instruments. Here, we show that the primary advantage of PRM is the ability to monitor all transitions in parallel and that high-resolution data are not necessary to obtain high-quality quantitative data. We run the same scheduled PRM assay, measuring 432 peptides from 126 plasma proteins, multiple times on an Orbitrap Eclipse Tribrid mass spectrometer, alternating separate liquid chromatography-tandem mass spectrometry runs between the high-resolution Orbitrap and the unit resolution linear ion trap for PRM. We find that both mass analyzers have similar technical precision and that the linear ion trap’s superior sensitivity gives it better lower limits of quantitation for over 62% of peptides in the assay.
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.
Data-independent acquisition (DIA) mass spectrometry has grown in popularity in recent years, because of the reproducibility and quantitative rigor of a systematic tandem mass spectrometry (MS/MS) ...sampling method. However, traditional DIA methods may spend valuable instrument time acquiring MS/MS spectra with no usable information in them, affecting sensitivity and quantitative performance. We developed a DIA strategy that dynamically adjusts the MS/MS windows during the chromatographic separation. The method focuses MS/MS acquisition on the most relevant mass range at each point in timeincreasing the quantitative sensitivity by increasing the time spent on each DIA window. We demonstrate an improved lower limit of quantification, on average, without sacrificing the number of peptides detected.
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.
Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ...ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.
We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer ...quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.
Infrared multiphoton dissociation (IRMPD) combined with ion trajectory simulations has been used to obtain probability maps of ion position as a function of different operating parameters in a ...quadrupole ion trap mass spectrometer. The factors that contribute to the depth of the pseudopotential trapping well are analyzed, and their effects on the efficiency of IRMPD are demonstrated. Ion trajectory simulations are used to substantiate experimental results and demonstrate in greater detail the dynamic nature of the ion population’s positional distribution. In particular, it is shown that the so-called “q z value” used during photodissociation can be of great consequence, as can the frequency of ac trapping voltage applied to the ring electrode. The results reveal that parameters which increase the pseudopotential well have the effect of decreasing the size of the ion cloud and maximizing overlap between the irradiating laser and the ions. Thus, while the common understanding of IRMPD dictates otherwise, IRMPD fragmentation efficiencies really depend on many ion trap operating parameters, much as collision-induced dissociation does.
We report the implementation of front-end higher energy collision-induced dissociation (fHCD) on a benchtop dual-pressure linear ion trap. Software and hardware modifications were employed, described ...in detail vide-infra, to allow isolated ions to undergo collisions with ambient gas molecules in an intermediate multipole (q00) of the instrument. Results comparing the performance of fHCD and resonance excitation collision-induced dissociation (RE-CID) in terms of injection time, total number of scans, efficiency, mass measurement accuracy (MMA), unique peptide identifications, and spectral quality of labile modified peptides are presented. fHCD is approximately 23% as efficient as RE-CID, and depending on the search algorithm, it identifies 6.6% more or 15% less peptides (q < 0.01) from a soluble whole-cell lysate (Caenorhabditis elegans) than RE-CID using Mascot or Sequest search algorithms, respectively. fHCD offers a clear advantage for the analysis of phosphorylated and glycosylated (O-GlcNAc) peptides as the average cross-correlation score (XCorr) for spectra using fHCD was statistically greater (p < 0.05) than for spectra collected using RE-CID.
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