Dataset Information

An IonStar Experimental Strategy for MS1 Ion Current-Based Quantification Using Ultrahigh-Field Orbitrap: Reproducible, In-Depth, and Accurate Protein Measurement in Large Cohorts.

ABSTRACT: In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar experimental strategy achieving excellent quantitative quality of MS1 quantification. Key features include: (i) an optimized, surfactant-aided sample preparation approach provides highly efficient (>75% recovery) and reproducible (<15% CV) peptide recovery across large cell/tissue cohorts; (ii) a long column with modest gradient length (2.5 h) yields the optimal balance of depth/throughput on a Q/UHF-Orbitrap; (iii) a large-ID trap not only enables highly reproducible gradient delivery as for the first time observed via real-time conductivity monitoring, but also increases quantitative loading capacity by >8-fold and quantified >25% more proteins; (iv) an optimized HCD-OT markedly outperforms HCD-IT when analyzing large cohorts with high loading amounts; (v) selective removal of hydrophobic/hydrophilic matrix components using a novel selective trapping/delivery approach enables reproducible, robust LC-MS analysis of >100 biological samples in a single set, eliminating batch effect; (vi) MS1 acquired at higher resolution (fwhm = 120 k) provides enhanced S/N and quantitative accuracy/precision for low-abundance species. We examined this pipeline by analyzing a 5 group, 20 samples biological benchmark sample set, and quantified 6273 unique proteins (?2 peptides/protein) under stringent cutoffs without fractionation, 6234 (>99.4%) without missing data in any of the 20 samples. The strategy achieved high quantitative accuracy (3-6% media error), low intragroup variation (6-9% media intragroup CV) and low false-positive biomarker discovery rates (3-8%) across the five groups, with quantified protein abundances spanning >6.5 orders of magnitude. Finally, this strategy is straightforward, robust, and broadly applicable in pharmaceutical/biomedical investigations.

SUBMITTER: Shen X

PROVIDER: S-EPMC5914162 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An IonStar Experimental Strategy for MS1 Ion Current-Based Quantification Using Ultrahigh-Field Orbitrap: Reproducible, In-Depth, and Accurate Protein Measurement in Large Cohorts.

Shen Xiaomeng X Shen Shichen S Li Jun J Hu Qiang Q Nie Lei L Tu Chengjian C Wang Xue X Orsburn Benjamin B Wang Jianmin J Qu Jun J

Journal of proteome research 20170525 7

In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar experimental strategy achieving excellent quantitative quality of MS1 quantification. Key ...[more]

PMID: 28412812

Similar Datasets

Project description:Robust, reliable quantification of large sample cohorts is often essential for meaningful clinical or pharmaceutical proteomics investigations, but it is technically challenging. When analyzing very large numbers of samples, isotope labeling approaches may suffer from substantial batch effects, and even with label-free methods, it becomes evident that low-abundance proteins are not reliably measured owing to unsufficient reproducibility for quantification. The MS1-based quantitative proteomics pipeline IonStar was designed to address these challenges. IonStar is a label-free approach that takes advantage of the high sensitivity/selectivity attainable by ultrahigh-resolution (UHR)-MS1 acquisition (e.g., 120-240k full width at half maximum at m/z = 200) which is now widely available on ultrahigh-field Orbitrap instruments. By selectively and accurately procuring quantitative features of peptides within precisely defined, very narrow m/z windows corresponding to the UHR-MS1 resolution, the method minimizes co-eluted interferences and substantially enhances signal-to-noise ratio of low-abundance species by decreasing noise level. This feature results in high sensitivity, selectivity, accuracy and precision for quantification of low-abundance proteins, as well as fewer missing data and fewer false positives. This protocol also emphasizes the importance of well-controlled, robust experimental procedures to achieve high-quality quantification across a large cohort. It includes a surfactant cocktail-aided sample preparation procedure that achieves high/reproducible protein/peptide recoveries among many samples, and a trapping nano-liquid chromatography-mass spectrometry strategy for sensitive and reproducible acquisition of UHR-MS1 peptide signal robustly across a large cohort. Data processing and quality evaluation are illustrated using an example dataset ( http://proteomecentral.proteomexchange.org ), and example results from pharmaceutical project and one clinical project (patients with acute respiratory distress syndrome) are shown. The complete IonStar pipeline takes ~1-2 weeks for a sample cohort containing ~50-100 samples.

Project description:Quantitative proteomics in large cohorts is highly valuable for clinical/pharmaceutical investigations but often suffers from severely compromised reliability, accuracy, and reproducibility. Here, we describe an ultra-high-resolution IonStar method achieving reproducible protein measurement in large cohorts while minimizing the ratio compression problem, by taking advantage of the exceptional selectivity of ultra-high-resolution (UHR)-MS1 detection (240k_FWHM@m/z = 200). Using mixed-proteome benchmark sets reflecting large-cohort analysis with technical or biological replicates (N = 56), we comprehensively compared the quantitative performances of UHR-IonStar vs a state-of-the-art SWATH-MS method, each with their own optimal analytical platforms. We confirmed a cutting-edge micro-liquid chromatography (LC)/Triple-TOF with Spectronaut outperforms nano-LC/Orbitrap for SWATH-MS, which was then meticulously developed/optimized to maximize sensitivity, reproducibility, and proteome coverage. While the two methods with distinct principles (i.e., MS1- vs MS2-based) showed similar depth-of-analysis (∼6700-7000 missing-data-free proteins quantified, 1% protein-false discovery rate (FDR) for entire set, 2 unique peptides/protein) and good accuracy/precision in quantifying high-abundance proteins, UHR-IonStar achieved substantially superior quantitative accuracy, precision, and reproducibility for lower-abundance proteins (a category that includes most regulatory proteins), as well as much-improved sensitivity/selectivity for discovering significantly altered proteins. Furthermore, compared to SWATH-MS, UHR-IonStar showed markedly higher accuracy for a single analysis of each sample across a large set, which is an inadequately investigated albeit critical parameter for large-cohort analysis. Finally, we compared UHR-IonStar vs SWATH-MS in measuring the time courses of altered proteins in paclitaxel-treated cells (N = 36), where dysregulated biological pathways have been very well established. UHR-IonStar discovered substantially more well-recognized biological processes/pathways induced by paclitaxel. Additionally, UHR-IonStar showed markedly superior ability than SWATH-MS in accurately depicting the time courses of well known to be paclitaxel-induced biomarkers. In summary, UHR-IonStar represents a reliable, robust, and cost-effective solution for large-cohort proteomic quantification with excellent accuracy and precision.

Project description:Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using fragment ions from MS/MS spectra to predict the structures of the unknown compounds. The precursor ion selected for fragmentation is commonly performed using data dependent acquisition (DDA) strategies or following statistical analysis using targeted MS/MS approaches. However, the selected precursor ions from DDA only cover a biased subset of the peaks or features found in full scan data. In addition, different statistical analysis can select different precursor ions for MS/MS analysis, which make the post-hoc validation of ions selected following a secondary analysis impossible for precursor ions selected by the original statistical method. Here we propose an automated, exhaustive, statistical model-free workflow: paired mass distance-dependent analysis (PMDDA), for reproducible untargeted mass spectrometry MS2 fragment ion collection of unknown compounds found in MS1 full scan. Our workflow first removes redundant peaks from MS1 data and then exports a list of precursor ions for pseudo-targeted MS/MS analysis on independent peaks. This workflow provides comprehensive coverage of MS2 collection on unknown compounds found in full scan analysis using a "one peak for one compound" workflow without a priori redundant peak information. We compared pseudo-spectra formation and the number of MS2 spectra linked to MS1 data using the PMDDA workflow to that obtained using CAMERA and RAMclustR algorithms. More annotated compounds, molecular networks, and unique MS/MS spectra were found using PMDDA compared with CAMERA and RAMClustR. In addition, PMDDA can generate a preferred ion list for iterative DDA to enhance coverage of compounds when instruments support such functions. Finally, compounds with signals in both positive and negative modes can be identified by the PMDDA workflow, to further reduce redundancies. The whole workflow is fully reproducible as a docker image xcmsrocker with both the original data and the data processing template.

Project description:BACKGROUND:Ultrasonography is a fast and patient-friendly modality to assess cartilage thickness. However, inconsistent results regarding accuracy have been reported. Therefore, we asked what are (1) the accuracy, (2) reproducibility, and (3) reliability of ultrasonographic cartilage thickness measurement using contrast-enhanced micro-CT for validation? METHODS:A series of 50 cartilage-bone plugs were harvested from fresh bovine and porcine joints. Ultrasonic cartilage thickness was determined using an A-mode, 20-MHz hand-held ultrasonic probe with native (1580?m/s) and adjusted speed of sound (1696?m/s). All measurements were performed by two observers at two different occasions. Angle of insonation was controlled by tilting the device and recording minimal thickness. Retrieval of exact location for measurement was facilitated by aligning the circular design of both cartilage-bone plug and ultrasonic device. There was no soft tissue interference between cartilage surface and ultrasonic probe. Ground truth measurement was performed using micro-CT with iodine contrast agent and a voxel size of 16??m. The mean cartilage thickness was 1.383?±?0.402?mm (range, 0.588-2.460?mm). RESULTS:Mean accuracy was 0.074?±?0.061?mm (0.002-0.256?mm) for native and 0.093?±?0.098?mm (0.000-0.401?mm) for adjusted speed of sound. Bland-Altman analysis showed no systematic error. High correlation was found for native and adjusted speed of sound with contrast-enhanced micro-CT (both r?=?0.973; p?<?0.001). A perfect agreement for reproducibility (intraclass correlation coefficient [ICC] 0.992 and 0.994) and reliability (ICC 0.993, 95% confidence interval 0.990-0.995) was found. CONCLUSIONS:Ultrasonic cartilage thickness measurement could be shown to be highly accurate, reliable, and reproducible. The A-mode ultrasonic cartilage thickness measurement is a fast and patient-friendly modality which can detect early joint degeneration and facilitate decision making in joint preserving surgery.

Project description: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.

Dataset Information

An IonStar Experimental Strategy for MS1 Ion Current-Based Quantification Using Ultrahigh-Field Orbitrap: Reproducible, In-Depth, and Accurate Protein Measurement in Large Cohorts.

Publications

An IonStar Experimental Strategy for MS1 Ion Current-Based Quantification Using Ultrahigh-Field Orbitrap: Reproducible, In-Depth, and Accurate Protein Measurement in Large Cohorts.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets