Highly Multiplexed Targeted Proteomics Acquisition on a TIMS-QTOF.

Native mass spectrometry (nMS) is an increasingly popular technique for studying intact protein quaternary structure. When coupled with ion mobility, which separates ions based on their size, charge, and shape, it provides additional structural information on the protein complex of interest. We present here data from a novel prototype TIMS (trapped ion mobility spectrometry)-quadrupole-SID (surface-induced dissociation)-time of flight, TIMS-Q-SID-TOF, instrument for nMS. The modifications include changing the TIMS cartridge from concave to convex electrode geometry with a dual TIMS tunnel design and operating TIMS at 425 kHz radio frequency (RF) to improve the trapping efficiency for high mass-to-charge (m/z) ion mobility analysis, such as 3 and 4 MDa hepatitis B virus capsids. The quadrupole RF driver was lowered to 385 kHz, which extends the isolation range from 3,000 to 17,000 m/z and allows isolation of a single charge state of GroEL at 16,200 m/z with an isolation window of 25 m/z. Finally, a 6 mm thick, 2-lens SID device replaced the collision cell entrance lens. SID dissociated 801 kDa GroEL into all combinations of subcomplexes, and the peaks were well-resolved, allowing for confident assignment of product ions. This is the first time a novel prototype timsTOF Pro for nMS has been introduced with high resolving power ion mobility separation coupled to high m/z quadrupole selection and SID for protein complex fragmentation with product ion collection and detection across a broad m/z range of 1,500 to 40,000.

The parallel accumulation-serial fragmentation (PASEF) approach based on trapped ion mobility spectrometry (TIMS) enables mobility-resolved fragmentation and a higher number of fragments in the same time period compared to conventional MS/MS experiments. Furthermore, the ion mobility dimension offers novel approaches for fragmentation. Using parallel reaction monitoring (prm), the ion mobility dimension allows a more accurate selection of precursor windows, while using data-independent aquisition (dia) spectral quality is improved through ion-mobility filtering. Owing to favorable implementation in proteomics, the transferability of these PASEF modes to lipidomics is of great interest, especially as a result of the high complexity of analytes with similar fragments. However, these novel PASEF modes have not yet been thoroughly evaluated for lipidomics applications. Therefore, data-dependent acquisition (dda)-, dia-, and prm-PASEF were compared using hydrophilic interaction liquid chromatography (HILIC) for phospholipid class separation in human plasma samples. Results show that all three PASEF modes are generally suitable for usage in lipidomics. Although dia-PASEF achieves a high sensitivity in generating MS/MS spectra, the fragment-to-precursor assignment for lipids with both, similar retention time as well as ion mobility, was difficult in HILIC-MS/MS. Therefore, dda-PASEF is the method of choice to investigate unknown samples. However, the best data quality was achieved by prm-PASEF, owing to the focus on fragmentation of specified targets. The high selectivity and sensitivity in generating MS/MS spectra of prm-PASEF could be a potential alternative for targeted lipidomics, e.g., in clinical applications.

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.

Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomolecules in tissues. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here, we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables a >250% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn backbone isomers, acyl chain isomers, and double-bond position and stereoisomers, is demonstrated. As a proof of concept, in situ separation and imaging of lipid isomers with distinct spatial distributions were performed using tissue sections from a whole-body mouse pup.

Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines an atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma postionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behavior of a variety of lipid classes in positive- and negative-ion modes, and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser postionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE), as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based postionization with ion mobility is presented, and we show the value of ion mobility for the resolution and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.

Theanalysis of complex biological mixtures remains a significantchallenge in mass spectrometry (MS), particularly when using conventionaldirect infusion MS/MS approaches due to inherent limitations in resolvingpower and spectral complexity. Here, we demonstrate the integrationof trapped ion mobility spectrometry (TIMS) with two-dimensional massspectrometry (2DMS) to enable high-resolution TIMS-MS/2DMS experimentsfor detailed protein characterization within mixtures. TIMS providesseparation based on the ion’s size-to-charge ratio, effectivelyreducing the occurrence of chimeric tandem mass spectra containingfragments from more than one precursor ion. This coupling allows foran improved peak capacity and reduced ambiguity in tandem spectralinterpretation. When applied to a model protein mixture, the TIMS-MS/2DMSmethod allows resolution of near m/z species, including isomeric and isonucleonic species, and it waspossible to assign secondary fragmentation with greater confidence.

PurposeProteomic composition of tears is not yet fully characterized and has a great potential to be used as source for discovery of disease‐specific protein biomarkers. The present work is a preliminary evaluation of tear proteome using timsTOF Pro®, a new highly sensitive mass spectrometry technology.Material and MethodsThree tear samples from 3 healthy subjects (HS) and 2 from a dry eye (DE) patient were collected using plastic micropipettes and Schirmer’s strips respectively. After protein elution from the strips, all samples were treated under the same conditions. Proteins were reduced and alkylated then digested in solution with LysC and Trypsin prior to nanoLC‐MS/MS analysis (nanoElute and timsTOF Pro®, Bruker). Protein identification was performed with Xtandem.ResultsIn total, 1191 proteins were identified in both analytical groups with at least 1 specific peptide: 446 proteins in HS and 1034 proteins in the DE; 802 proteins were exclusively identified in DE (e.g. A2M, HPX, ALDH3A1, PKM, MYH14, ALDH3A1) while 157 were expressed only in controls (e.g. PLOD1, DNAJC3, GOLM1, RNASE4, SDF4, HYOU1). In DE cell structural associated proteins as well as proteins highly correlated with inflammatory and apoptotic processes were detected. Finally, among the proteins that are common to both groups 122 were upregulated in DE vs HS (e.g. ALB, TF, ENO1, ANXA1, GSTP1, S100A9) while 88 were downregulated (e.g. LTF, LCN1, LPO, CST4, LACRT, AZGP1). The upregulated proteins are associated with a blood‐lachrymal barrier failure, inflammatory & immune responses while the downregulated ones sign a lachrymal gland dysfunction.ConclusionsDifferences in the protein expression between HS and DE arise due to the pathological conditions but also due to the different sampling methods used. This preliminary study could offer a roadmap for designing further studies for the in‐depth characterization of the tear protein profile expression under physiological and pathological conditions.

Protein post-translational modifications provide critical proteomic details towards elucidating mechanisms of altered protein function due to toxic exposure, altered metabolism, or disease pathogenesis. Lysine propionylation is a recently described modification that occurs due to metabolic alterations in propionyl-CoA metabolism and sirtuin depropionylase activity. Acrolein is a toxic aldehyde generated through exogenous and endogenous pathways, such as industrial exposure, cigarette smoke inhalation, and non-enzymatic lipid peroxidation. Importantly, lysine modifications arising from propionylation and acroleination can be isobaric - indistinguishable by mass spectrometry - and inseparable via reverse-phase chromatography. Here, we present the novel application of trapped ion mobility spectrometry (TIMS) to resolve such competing isobaric lysine modifications. Specifically, the PTM products of a small synthetic peptide were analyzed using a prototype TIMS - time-of-flight mass spectrometer (TIMS-TOF). In that the mobilities of these propionylated and acroleinated peptides differ by only 1%, a high-resolution mobility analysis is required to resolve the two. We were able to achieve more than sufficient resolution in the TIMS analyzer (~170), readily separating these isobars.

BackgroundLipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. ResultsA SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SignificanceThis method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

The sequencing of intact proteins within a mass spectrometer has many benefits but is frequently limited by the fact that tandem mass spectrometry (MS/MS) techniques often generate poor sequence coverages when applied to protein ions. To overcome this limitation, exotic MS/MS techniques that rely on lasers and radical chemistry have been developed. These techniques generate high sequence coverages, but they require specialized instrumentation, create products through multiple dissociation mechanisms, and often require long acquisition times. Recently, we demonstrated that protein ions can be dissociated in a trapped ion mobility spectrometry (TIMS) device prior to mobility separation in a commercial timsTOF. All generated product ions were distributed throughout the mobility dimension, and this separation enabled deconvolution of complex tandem mass spectra and could enable facile pseudo-MS3 interrogation of generated product ions with the downstream quadrupole and collision cell. A second activation step improves sequence coverage because the most labile bonds have been depleted during the first dissociation and subsequent dissociation events are more evenly distributed throughout the product ion backbone. In this work, we explore the potential of this mobility-assisted pseudo-MS3 (MAP) method on a commercial timsTOF and timsTOF Pro 2. We demonstrate that while MAP only generates 92% of the sequence coverage of the most effective MS/MS technique, it accomplished this feat in 1.5 min and could be facilely integrated with liquid chromatographic separations.

Trends in trapped ion mobility – Mass spectrometry instrumentation

The separation of isomeric compounds with major differences in their physiochemical and pharmacokinetic properties is of particular importance in pharmaceutical R&D. However, the structural assessment and separation of these compounds with current analytical techniques and methods are still a challenge. In this study, we describe strategies to separate the various structural and stereo-isomers. The separation of ten structural and stereo-isomers was investigated using Trapped and Travelling Wave ion mobility spectrometry (TIMS and TWIMS). Different strategies including adduct ion formation with Na, Li, Ag and Cs as well as fragmentation before and after the ion mobility cell were applied to separate the isomeric compounds. All the counter ions (in particular Na) strongly coordinated with the test analytes in all the IMS systems. The highest resolving power was achieved for the sodium and lithium adducts using TIMS-time-of-flight (TOF). However, some separation was attained on a Synapt HDMS system with its unique potential to monitor the ion mobility of the product ions. The elution order of the adduct ions was the same in all instruments, in which, unexpectedly, the para-substituted isomer of the [M + Na]+ species had the lowest collision cross section followed by the meta- and ortho-isomers. The formation of adduct ions could facilitate the separation of structural and even stereo-isomers by generating different molecular conformations. In addition, fragmenting isomers before or after the ion mobility cell is a valuable strategy to separate and also to assess the structures of adducts and different conformers.

The complexity of biological matrices makes the detection and quantification of compounds of interest challenging. For successful targeted or untargeted identification of compounds within a biological environment, the use of complementary separation techniques is routinely required; in many situations, a single analytical technique is not sufficient. In the present dissertation, a multidimensional analytical technique was developed and evaluated, a combination of new sample preparation/extraction protocols, liquid chromatography, trapped ion mobility and mass spectrometry (e.g., LC-TIMS-MS and LC-TIMS-MS/MS). The performance of these techniques was evaluated for the detection of polybrominated diphenyl ethers metabolites, polychlorinated biphenyls metabolites in human plasma, opioid metabolites in human urine, and lipids in Dictyostelium discoideum cells. The new workflows and methods described in the body of this dissertation allows for rapid, selective, sensitive and high-resolution detection of biomarkers in biological matrices with increased confidence, sensitivity and shorter sample preparation and analysis time.

Combining trapped ion mobility spectrometry with liquid chromatography and tandem mass spectrometry for analysis of isomeric PDE-5 inhibitor analogs