Multiplexed quantitation strategies using Thermo ScientificTM Tandem Mass TagsTM (TMTTM) enable precise measurement of peptide or protein abundance from multiple samples into a single highresolution LC-MS analysis. Increasingly, various biological experiments demand higher quantitation accuracy and proteome coverage. Here, we demonstrate increased quantitative performance and throughput efficiency on a Thermo ScientificTM Orbitrap ExplorisTM 480 mass spectrometer combined with a Thermo ScientificTM FAIMS ProTM interface.

Technology, Cambridge, MA Aaron S. Gajadhar1, Lauren E. Stopfer2, Cameron T. Flower2, Forest M. White2, Bhavin Patel3, Sebastien Gallien4, Romain Huguet1, Graeme McAlister1, Derek Bailey1, Shannon Eliuk1, Markus Kellmann5, Tabiwang N. Arrey5, Alexander Harder5, Daniel Lopez-Ferrer1, Andreas Huhmer1. Thermo Fisher Scientific, San Jose1, CA, USA; Rockford3, IL, USA; PMSC4, Cambridge, MA, USA; Bremen, Germany5; Department of Biological Engineering2, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA

Quantitative bottom-up shotgun lipidomics relies on molecular species-specific "signature" fragments consistently detectable in tandem mass spectra of analytes and standards. Molecular species of glycerophospholipids are typically quantified using carboxylate fragments of their fatty acid moieties produced by higher-energy collisional dissociation of their molecular anions. However, employing standards whose fatty acids moieties are similar, yet not identical, to the target lipids could severely compromise their quantification. We developed a generic and portable fragmentation model implemented in the open-source LipidXte software that harmonizes the abundances of carboxylate anion fragments originating from fatty acid moieties having different sn-1/2 positions at the glycerol backbone, length of the hydrocarbon chain, and number and location of double bonds. The postacquisition adjustment enables unbiased absolute (molar) quantification of glycerophospholipid species independent of instrument settings, collision energy, and employed internal standards.

Olaf Scheibner,1 Maciej Bromirski,1 Markus Kellmann,1 Sebastian Westrup,2 Charles Yang3 1Thermo Fisher Scienti c, Bremen, Germany, 2Thermo Fisher Scienti c, Dreieich, Germany, 3Thermo Fisher Scienti c, San Jose, CA, USA P o ster N o te 16 6 7 Quick and Sensitive Analysis of Multiclass V terinary Drug Residues in Animal Products Using a Novel Benchtop Orbitrap Mass Spectrometry Syste Olaf Scheibner1, Maciej Bro irski1, Markus Kellmann1, Sebastian Westrup2, Charles Yang3 Thermo Fisher Scientific, Bremen, Germany1, Thermo Fisher Scientific, Dreieich, Germany2, Thermo Fisher Scientific, San Jose, CA, USA3

Goal Forty-four multi-class veterinary drugs of known concentrations were employed to demonstrate how the use of a generic variable dataindependent acquisition (vDIA) method with wide MS/MS precursor isolation windows achieves sensitivity and selectivity comparable to data-dependent MS2 acquisition (using narrow isolation windows) in quantitative and qualitative small molecule applications. In addition, a full record of MS and MS/MS data for the measured sample fit for non-targeted and unknown screening purposes is delivered. setup, five isolation windows are set, covering the entire mass range of the preceding full scan. Figure 1 diagrams a representative vDIA method setup. Figure 1B shows an alternative setup, covering a wider mass range. Up to eight isolation windows can be used to span the full-scan mass range. However, earlier studies indicate that five fragmentation windows represent an optimal compromise between sensitivity and selectivity on the one hand and scan speed on the other.1

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 μg of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.

In mass spectrometry-based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold.

Each scan isolates and fragments five 4 m/z – wide precursor ranges randomly chosen from a predefined list of consecutive, adjacent ranges. In this example, the mass range from 500-900 m/z is sampled every 20 scan events. Step 1: Generate a list of isolation windows to be sampled (in this example, 100 4 m/z wide windows 500-900 m/z) Step 2: Build a scan by removing 5 isolation windows at random from the isolation window list, repeat until the isolation window list is empty Step 3: Repopulate the isolation window list, go to step 2. Q-Exactive Isolation Window Uniformity

In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ9-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0–20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80–120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

Introduction Mass spectrometry has become an essential tool for understanding aspects of biological systems such as proteome dynamics and signaling regulation.1 When coupled with multi-dimensional liquid chromatography, modern mass spectrometers, such as the Thermo Scientific LTQ Orbitrap Velos hybrid ion trap-Orbitrap MS, are capable of identifying thousands of peptides and posttranslation modifications per hour on a routine basis. Relative quantitation, through a label-free approach or via incorporation of heavy isotopes such as SILAC2 or isobaric tagging (TMT)3, enables the identification of candidates displaying biologically interesting dynamics on a global scale in the early discovery phase. It is now common to find several hundred biomarker candidates, including many novel ones, in a complex biological system. Putative biomarkers can also be generated through hypothesis or via literature knowledge. Putative biomarkers need to be quantitatively analyzed across large numbers of samples from multiple biological sources and conditions, either for understanding of signaling regulation or for verification and selection of final biomarkers. A fast, robust and cost-effective platform is required at this verification stage. The presence of large numbers of diverse candidates makes it impractical to use traditional antibody-based immunoassays. A targeted MS approach, in particular selected reaction monitoring (SRM), has become the preferred platform for quantitative analysis of tens to hundreds of peptide candidates.4,5 Detection of target proteins at low micrograms per liter has been reported from depleted human plasma using SRM.6,7 SRM approaches on triple-quadrupole mass spectrometers can be very cost-effective, have higher selectivity, and be easier to develop than traditional ELISAs, yet several major challenges remain: • The balance between duty cycle and sensitivity limits the number of peptide targets that can be analyzed in a single experiment. This problem can be partially solved by incorporating retention time windows. • The low resolution of quadrupoles limits their ability to distinguish targets from complex backgrounds. As a result, the detection limit is often in the low milligrams per liter in untreated plasma samples. • The development of an SRM-based assay for a given protein is time consuming. It has been estimated that only ~100 such assays could be developed over a year even in an expert laboratory.8 • Some peptide targets, especially those with higher charge states, higher mass, or modifications, do not fragment well and are therefore difficult to analyze by SRM. This poses a major problem for many functional-oriented analyses because there are no alternative peptide targets to select.

本文概述了基于具有卓越性能的Thermo Scientific Orbitrap静电场轨道阱质谱仪的新型Exactive台式质谱仪,对其主要的特性包括仪器设计、分析参数及应用进行了介绍.

Background: Today, dementias are diagnosed late in the course of disease. Future treatments have to start earlier in the disease process to avoid disability requiring new diagnostic tools. The objective of this study is to develop a new method for the differential diagnosis and identification of new biomarkers of Alzheimer's disease (AD) using capillary-electrophoresis coupled to mass-spectrometry (CE-MS) and to assess the potential of early diagnosis of AD.Methods and Findings: Cerebrospinal fluid (CSF) of 159 out-patients of a memory-clinic at a University Hospital suffering from neurodegenerative disorders and 17 cognitively-healthy controls was used to create differential peptide pattern for dementias and prospective blinded-comparison of sensitivity and specificity for AD diagnosis against the Criterion standard in a naturalistic prospective sample of patients. Sensitivity and specificity of the new method compared to standard diagnostic procedures and identification of new putative biomarkers for AD was the main outcome measure. CE-MS was used to reliably detect 1104 low-molecular-weight peptides in CSF. Training-sets of patients with clinically secured sporadic Alzheimer's disease, frontotemporal dementia, and cognitively healthy controls allowed establishing discriminative biomarker pattern for diagnosis of AD. This pattern was already detectable in patients with mild cognitive impairment (MCI). The AD-pattern was tested in a prospective sample of patients (n = 100) and AD was diagnosed with a sensitivity of 87% and a specificity of 83%. Using CSF measurements of beta-amyloid1-42, total-tau, and phospho(181)-tau, AD-diagnosis had a sensitivity of 88% and a specificity of 67% in the same sample. Sequence analysis of the discriminating biomarkers identified fragments of synaptic proteins like proSAAS, apolipoprotein J, neurosecretory protein VGF, phospholemman, and chromogranin A.Conclusions: The method may allow early differential diagnosis of various dementias using specific peptide fingerprints and identification of incipient AD in patients suffering from MCI. Identified biomarkers facilitate face validity for the use in AD diagnosis.

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Current approaches to discovery-stage drug metabolism studies (pharmacokinetics, microsomal stability, etc.) typically use triple-quadrupole-based approaches for quantitative analysis. This necessitates the optimization of parameters such as Q1 and Q3 m/z values, collision energy, and interface voltages. These studies detect only the specified compound and information about other components, such as metabolites, is lost. The ability to perform full-scan acquisition for quantitative analysis would eliminate the need for compound optimization while enabling the detection of metabolites and other non-drug-related endogenous components. Such an instrument would have to provide sensitivity, selectivity, dynamic range, and scan speed suitable for discovery-stage quantitative studies. In this study, a prototype benchtop Orbitrap-based mass analyzer was used to collect both quantitative and qualitative data from human microsomal incubation samples as well as rat plasma from pharmacokinetic studies. Instrumental parameters such as scan speed, resolution, and mass accuracy are discussed in relation to the requirements for a quantitative-qualitative workflow. The ability to perform highly selective quantitative analysis while simultaneously characterizing metabolites from both in vitro and in vivo studies is discussed.

In LC full scan based MS screening methods correct mass assignment is essential. Parameters affecting the accuracy of mass assignment, i.e., analyte concentration, complexity of the matrix, and resolving power, were studied using typical examples from the field of residue and contaminant analysis in food and feed. The evaluation was carried out by analyzing samples of honey and animal feed, spiked with 151 pesticides, veterinary drugs, mycotoxins, and plant toxins at levels ranging from 10 to 250 ng/g. Analyses were performed using a single stage Orbitrap with resolving power settings varying from 10,000 to 100,000 (FWHM). For consistent and reliable mass assignment (<2 ppm) of analytes at low levels in complex matrices, a high resolving power (≥50,000) was found to be required. At lower resolving power settings, the error in the assignment of mass increased due to the coelution of analytes with interferences at the same nominal mass. This negatively affected selectivity and quantitative performance due to the inability to use the required narrow mass-extraction windows. In the case of the less complex honey matrix, a resolving power of 25,000 was generally sufficient to obtain a mass assignment error close to the typical instrument mass accuracy (≤2 ppm) down to low concentration levels of 10 ng/g.

Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a "training set" for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a "test set." The signature panel showed sensitivity of 98% (95% confidence interval, 88.7-99.6) and 83% specificity (95% confidence interval, 51.6-97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment.