The ion mobility enhanced MaxQuant software is introduced, offering an end to end computational workflow for LC-IMS-MS/MS shotgun proteomics data. Highly parallelizable 4D feature detection and ...de-isotoping algorithms are featured and non-linear multi-dimensional mass recalibration is performed. A new matching between runs (MBR) algorithm that utilizes aligned peptide collisional cross sections (CCS). MS1 level label-free quantification is also implemented providing good results. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.
Display omitted
Highlights
•Highly parallelizable 4D feature detection in ion mobility enhanced shotgun proteomics.•Multidimensional non-linear mass, retention time and ion mobility recalibration.•Collision cross section aware matching between runs.•Label-free quantification of ion mobility MS data.
Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range. Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides and proteins in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In liquid chromatography-mass spectrometry (LC-MS)-based proteomics, many precursors elute from the column simultaneously. In data-dependent analyses, these precursors are fragmented one at a time, ...whereas the others are discarded entirely. Here we employ trapped ion mobility spectrometry (TIMS) on an orthogonal quadrupole time-of-flight (QTOF) mass spectrometer to remove this limitation. In TIMS, all precursor ions are accumulated in parallel and released sequentially as a function of their ion mobility. Instead of selecting a single precursor mass with the quadrupole mass filter, we here implement synchronized scans in which the quadrupole is mass positioned with sub-millisecond switching times at the m/z values of appropriate precursors, such as those derived from a topN precursor list. We demonstrate serial selection and fragmentation of multiple precursors in single 50 ms TIMS scans. Parallel accumulation–serial fragmentation (PASEF) enables hundreds of MS/MS events per second at full sensitivity. Modeling the effect of such synchronized scans for shotgun proteomics, we estimate that about a 10-fold gain in sequencing speed should be achievable by PASEF without a decrease in sensitivity.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Single‐cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in‐depth ...characterization of individual cells by mass spectrometry (MS)‐based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow‐rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10‐fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS‐isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single‐cell proteomes to transcriptome data revealed a stable‐core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra‐high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.
Synopsis
A new ultra‐high sensitivity LC‐MS workflow increases sensitivity by up to two orders of magnitude and enables true single‐cell proteome analysis. In‐depth comparison indicates that the single‐cell transcriptome is stochastic while the single‐cell proteome is complete and stable.
A highly optimized data independent acquisition powered single‐cell proteomics workflow including sub‐µl sample preparation, very low flow chromatography and trapped ion mobility mass spectrometry (diaPASEF) is presented.
Single‐cell proteome analysis is performed by injecting cells one‐by‐one across the cell cycle into the LC‐MS and correctly identifies cell states.
Single‐cell proteome information is highly complementary to single‐cell transcriptome information.
At the single‐cell level the proteome is quantitatively and qualitatively stable, while the transcriptome is stochastic.
A new ultra‐high sensitivity LC‐MS workflow increases sensitivity by up to two orders of magnitude and enables true single‐cell proteome analysis. In‐depth comparison indicates that the single‐cell transcriptome is stochastic while the single‐cell proteome is complete and stable.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Trapped ion mobility spectrometry: A short review Ridgeway, Mark E.; Lubeck, Markus; Jordens, Jan ...
International journal of mass spectrometry,
February 2018, 2018-02-00, Volume:
425
Journal Article
Peer reviewed
Display omitted
•TIMS is an ion funnel based analyzer design which blends small size, high resolving power, and high sensitivity.•TIMS operates on the same principles as much larger traditional drift ...tube ion mobility analyzers.•The flexibility of TIMS lends itself to a number of applications and hardware designs.•TIMS flexibility allows for the effective hybridization of TIMS with TOF and FT-ICR mass analyzers.
Trapped ion mobility spectrometry (TIMS) hybridized with mass spectrometry (MS) is a relatively recent advance in the field of ion mobility mass spectrometry (IMMS). The basic idea behind TIMS is the reversal of the classic drift cell analyzer. Rather than driving ions through a stationary gas, as in a drift cell, TIMS holds the ions stationary in a moving column of gas. This has the immediate advantage that the physical dimension of the analyzer can be small (∼5 cm) whereas the analytical column of gas – the column that flows past during the course of an analysis – can be large (as much as 10 m) and user defined. In the years since the first publication, TIMS has proven to be a highly versatile alternative to drift tube ion mobility achieving high resolving power (R ∼ 300), duty cycle (100%), and efficiency (∼80%). In addition to its basic performance specifications, the flexibility of TIMS allows it to be adapted to a variety of applications. This is highlighted particularly by the PASEF (parallel accumulation serial fragmentation) workflow, which adapts TIMS-MS to the shotgun proteomics application. In this brief review, the general operating principles, theory, and a number of TIMS-MS applications are summarized.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Data-independent acquisition (DIA) methods have become increasingly popular in mass spectrometry–based proteomics because they enable continuous acquisition of fragment spectra for all precursors ...simultaneously. However, these advantages come with the challenge of correctly reconstructing the precursor–fragment relationships in these highly convoluted spectra for reliable identification and quantification. Here, we introduce a scan mode for the combination of trapped ion mobility spectrometry with parallel accumulation—serial fragmentation (PASEF) that seamlessly and continuously follows the natural shape of the ion cloud in ion mobility and peptide precursor mass dimensions. Termed synchro-PASEF, it increases the detected fragment ion current several-fold at sub-second cycle times. Consecutive quadrupole selection windows move synchronously through the mass and ion mobility range. In this process, the quadrupole slices through the peptide precursors, which separates fragment ion signals of each precursor into adjacent synchro-PASEF scans. This precisely defines precursor–fragment relationships in ion mobility and mass dimensions and effectively deconvolutes the DIA fragment space. Importantly, the partitioned parts of the fragment ion transitions provide a further dimension of specificity via a lock-and-key mechanism. This is also advantageous for quantification, where signals from interfering precursors in the DIA selection window do not affect all partitions of the fragment ion, allowing to retain only the specific parts for quantification. Overall, we establish the defining features of synchro-PASEF and explore its potential for proteomic analyses.
Display omitted
•Synchro-PASEF is a highly efficient scan mode for MS-based proteomics.•It allows very fast cycle times, amplifying the fragment signal three-fold.•Fragments are directly linked to precursors via precursor slicing.•Precursor slicing enables deconvolution to pure fragmentation spectra.•Synchro-PASEF unites the benefits of data-dependent and data-independent acquisition.
The novel scan mode synchro-PASEF efficiently follows the natural shape of the precursor cloud in the m/z and the trapped ion mobility space. This manifests in short cycle times and high sampling frequency of the eluting peptides and fragment signal amplification. Additionally, the seamlessly movement of the quadrupole nearly universally slices the precursor in the ion mobility dimension. The slicing position adds tremendous specificity, allowing deconvolution of the fragment space to ‘pure fragmentation spectra’ reminiscent of data-dependent acquisition spectra.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to ...perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.
Synopsis
The study introduces µPhos, an accessible, plate-based workflow for low-input mass spectrometry-based phosphoproteomics. µPhos greatly facilitates high-dimensional experimental designs, as demonstrated by a 96-well drug response screen and analysis of small anatomical regions in the mouse brain.
Systematic optimization results in sensitive and selective enrichment of phosphopeptides in <100 µL, allowing direct processing of 96-well plates within one working day.
µPhos combined with dia-PASEF reproducibly quantifies 6000 to 17,000 confidently localized phosphosites from 1 to 20 µg protein starting material.
Sub-region resolved analysis of the mouse brain phosphoproteome infers spatially distinct kinase activity.
Time-course analysis of chronic myeloid leukemia cells treated with tyrosine kinase inhibitors reveals drug-specific responses.
The study introduces µPhos, an accessible, plate-based workflow for low-input mass spectrometry-based phosphoproteomics. µPhos greatly facilitates high-dimensional experimental designs, as demonstrated by a 96-well drug response screen and analysis of small anatomical regions in the mouse brain.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
PASEF multiplies the sequencing speed without any loss in sensitivity and is implemented in the timsTOF Pro instrument introduced here. Sequencing speeds above 100 Hz enable single run proteome ...analysis at a depth of 6000 proteins, making the instrument particularly attractive for rapid and highly sensitive proteomics. Collisional cross sections can be determined with up to 0.1% precision and acquired on a scale of 100,000s, which opens exciting areas for proteomics exploration.
Display omitted
Highlights
•Online PASEF achieves greater than 100 MS/MS per second at full sensitivity.•Accurate label-free quantification of over 6000 proteins in 2 h.•High throughput demonstrated on 50 ng digests measured in 5 min.•High-precision determination of 100,000 peptide collisional cross sections.
In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection ...windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.
Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in ...terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum−the highest proteome coverage reported with a QTOF instrument so far.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) ...series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon–stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma– and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.
Display omitted
•Using ion mobility as a glycoproteomics acquisition routine on the timsTOF Pro.•Optimization of collisional energies for glycopeptide fragmentation.•Ion-mobility–based focusing of instrument time on glycopeptides.•Increasing data quality by combining duplicate spectra generated by instrument speed.•Glycoproteomics analyses on high-complexity samples (human plasma and neutrophils).
In this study, we advance the use of the timsTOF Pro by designing and applying a novel acquisition routine for glycoproteomics. We demonstrate that the instrument can be specifically focused on sequencing glycopeptides by making use of ion mobility, and that its high scan rate can be used to improve spectrum quality. This can be applied to great effect as we show that, especially for shorter gradients, the number of glycopeptide detections can be sustained at a high level.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
