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Label-Free Quantitation
Explore the proteome in greater depth
As a cost-effective alternative to isotopic labeling approaches, label-free quantitation (LFQ) enables relative quantitation of protein samples from any origin. Samples are tested individually using advanced software with chromatographic features that align the various runs. The biggest advantage of this approach is that the number of sample comparisons is not limited. Additionally, peptide identification can be performed with any fragmentation method (CID, ETD, EThcD, and/or HCD).
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Improve DIA performance on Orbitrap mass spectrometers
Improve DIA performance on Orbitrap mass spectrometers
Access the Velocity DIA information kit, infographic, and other tools to enhance DIA performance on Orbitrap mass spectrometers.
Access important DIA tools ›
Faster throughput with greater proteome coverage for LFQ
The Thermo Scientific Orbitrap Astral Mass Spectrometer combines the proven performance of Orbitrap technology with the speed (200 Hz) and sensitivity of the novel Astral mass analyzer, enabling faster throughput and deeper proteome coverage in label-free quantitation (LFQ) experiments . The mass spectrometer enables analysis of thousands of samples without loss in performance, delivers accurate and reproducible quantitation of thousands of proteins.
Faster throughput with greater proteome coverage for LFQ
The Orbitrap Astral mass spectrometer combines the proven performance of Orbitrap technology with the speed (200 Hz) and sensitivity of the novel Astral mass analyzer, enabling faster throughput and deeper proteome coverage in label-free quantitation (LFQ) experiments . The mass spectrometer enables analysis of thousands of samples without loss in performance, delivers accurate and reproducible quantitation of thousands of proteins.
Label-free relative quantitation instruments
 Orbitrap Ascend Tribrid Mass Spectrometer |  Orbitrap Exploris 480 Mass Spectrometer |  Orbitrap Exploris 240 Mass Spectrometer |  Orbitrap Astral Mass Spectrometer | |
|---|---|---|---|---|
| Advantages | Obtain maximum insights on complex samples. Facilitate advanced proteomics workflows: label-free quantitation, whole proteome profiling and single-cell proteomics with faster data acquisition. Orbitrap Ascend Mass Spectrometer’s new innovations deliver ultimate flexibility in experimental scope, accelerating your path to new, impactful results, to drive your science beyond today’s discovery. | Get unparalleled insights from untargeted proteome profiles to targeted proteomics experiments with industry-leading single-cell sensitivity and accuracy. Curated workflows accelerate large-scale studies, delivering high-quality data and time savings for actionable outcomes. | Optimized proteomics solutions for small-to-large-scale studies with fast turnaround times and best-in-class performance in a compact footprint. From protein identification to multiplexing, expand your capabilities with everyday versatility. | By combining the proven performance of the Orbitrap mass analyzer with the speed (200 Hz) and sensitivity of the novel Astral mass analyzer, the Orbitrap Astral mass spectrometer enables faster throughput and deeper proteome coverage. |
| Resolving Power | Up to 480,000 (FWHM) at m/z 200 | Up to 480,000 at m/z 195 | Up to 240,000 FWHM at m/z 200 | Orbitrap resolution: settings range from 7500 to 480,000 (FWHM) at m/z 200 Astral resolution: 80,000 (FWHM) at m/z 524 |
| Scan Speed | Orbitrap mass analyzer MSⁿ up to 45 Hz Ion trap MSⁿ up to 50 Hz | Up to 40Hz | Up to 40Hz | Orbitrap MSn acquisition rates up to 40 Hz Astral MS2 acquisition rates up to 200 Hz |
| Mass Range | Standard mass range m/z 40–2000, mid-mass range m/z 200–6000, and optional HMRn+ mass range m/z 500–16,000 | 40 to 6,000 m/z (up to 8,000 m/z with the BioPharma option) | 40 to 6,000 m/z (up to 8,000 m/z with the BioPharma option) | Orbitrap: Standard mass range m/z 40–6000, m/z 40–8000 with BioPharma option Astral: Standard mass range m/z 40–6000 |
| Dynamic Range | >5000 with a single OTMS spectrum | >5000:1 | >5000:1 | Orbitrap: >5000 within a single Orbitrap mass spectrum Astral: >1000 within a single Astral mass spectrum |
| Mass Accuracy | Internal <1 ppm RMS; External: <3 ppm RMS | Internal <1 ppm RMS; External: <3 ppm RMS | Internal <1 ppm RMS; External: <3 ppm RMS | Orbitrap: Internal <1 ppm RMS; External: <3 ppm RMS Astral: External <3 ppm RMS |
Obtain broad peptide coverage
During label-free relative quantitation workflows, digested peptides are separated by LC prior to MS. RP separation on Thermo Scientific low-flow systems integrates well with label-free quantitation workflows and combines seamlessly with Thermo Scientific Orbitrap mass spectrometers. The Thermo Scientific Vanquish Neo UHPLC System is the system of choice. The Vanquish Neo UHPLC System combines an unrivaled degree of innovation to deliver 24/7 reproducible separations of complex mixtures at maximum performance for a variety of high-sensitivity LC-MS workflows
The Thermo Scientific FAIMS Pro Duo interface enhances precursor selectivity, improving qualitative and quantitative results for most peptide and protein applications while reducing time-consuming sample preparation steps. The FAIMS Pro Duo interface increases analytical performance through gas-phase fractionation and selective enhancement of peptidic compounds, reducing the complexity of the MS spectra, and improving the analyte signal-noise ratio. The end result is greater proteome coverage, enhanced sensitivity and more accurate quantitation.
Software for label-free quantitation
Discover novel protein biomarkers
Thermo Scientific Proteome Discoverer Software, with its new label-free quantitation node, provides an easy-to-use automated solution for evaluating large amounts of LC-MS data generated by label-free experiments. The label-free quantitation node also features a full suite of quantitative capabilities previously only available for isotopically labeled workflows. The combination of label-free quantification workflows with the scaling, normalization, and study management features of Proteome Discoverer software offers powerful means for analyzing highly complex data.
Label-free quantitation workflow
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Two different LFQ approaches to identification data acquisition
In a LFQ DDA approach, the ions for a given m/z range are individually isolated and fragmented. The quantitation involves extracting peptide chromatograms (MS
In traditional data-dependent workflows, consistent precursor and thereby protein quantitation can be challenging to achieve due to the stochastic sampling nature of the mass spectrometer. Innovations in software algorithms now extract LC-MS peaks in the raw data files and map them to identified spectra, detecting these features across LC-MS data files using retention-time alignment and feature linking, minimizing "missing data points" and maximizing quantitative insights.
In a LFQ DIA approach, a precursor mass range is selected and then divided into a series of relatively wide isolation windows: for example, 25 m/z each. Chimeric MS/MS data is acquired from all detected precursor ions in the first isolation window and then repeated for each consecutive, adjacent isolation window until the entire precursor mass range is covered. MS/MS spectral libraries are used to identify peptides of interest from the acquired data.
DIA significantly increases coverage and reproducibility by acquiring MS/MS data from all detected precursor ions; it also makes retrospective data analysis possible. LFQ DIA involves extracting peptide chromatograms (MS
Velocity LFQ DIA workflow on an Orbitrap Exploris 480 mass spectrometer
Learn about the qualitative and quantitative performance of the Velocity LFQ DIA workflow on an Orbitrap Exploris 480 MS for large-scale proteomics analysis.
Velocity LFQ DIA workflow on an Orbitrap Ascend Tribrid mass spectrometer
Learn about the qualitative and quantitative performance of the Velocity LFQ DIA workflow on an Orbitrap Ascend Tribrid MS for large-scale proteomics analysis.
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