Advanced FTMS Data Acquisition and Processing

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FTMS Booster : New Generation ElectronicsMeasures transients from any FTMS instrumentImproves performance and productivity of FTMS instrumentsEnables advanced diagnostics and workflow optimization

Key features of FTMS Booster technology :• Hardware interface to any FTMS instrument, for example LTQ FT series, LTQ OrbitrapTM, OrbitrapTM FusionTM, and

QExactiveTM OrbitrapTM series FTMS instruments, as well as SolariXTM FT-ICR MS instruments;• Works in parallel with manufacturer data acquisition software, e.g., XCaliburTM.• Real-time visualization of transients and mass spectra with or without data summation.• High-resolution and sensitivity (dynamic range) due to low-noise RF front-end electronics and high-throughput

FPGA with on-the-fly digital signal processing.• Full transient acquisition and pure absorption-mode FT on OrbitrapTM FTMS platforms and absorption-mode or

magnitude-mode FT on FT-ICR MS platforms.• Rapid data transfer for large data sets (spectra in full profile mode), high duty cycle (fast FTMS instruments), or

continuous recording of long transients (> 50 s).• Open file format : HDF5 (transients or mass spectra).• High-capacity (2 TB), fast SSD buffer for transients and mass spectra, with on-line interface for streaming to the

customer’s file server(s).

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Advanced FTMS WorkflowsThe Spectroswiss software solutions Peak-by-Peak and AutoVectis, alone or with our high-performance data acquisition system FTMS Booster, provide a quantum boost in FTMS data quality and processing sophistication, helping you to take on the most complex and challenging applications, including petroleomics :

FTMSBooster

FTMSinstruments

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AutoVectis : Absorption Mode FTProcessing FTMS spectra in absorption mode, instead of the normal magnitude mode, lets you detect more peaks and gives you improved mass accuracy. Or it lets you record spectra up to twice as fast. Also, as it is a post-processing technique, it does not require any changes to the instrument. Using powerful algorithms, AutoVectis automates the steps required to produce absorption mode FT (aFT) mass spectra so the benefits can be easily enjoyed by everyone.

Key features of AutoVectis :• Easy to learn, absorption mode Fourier transform signal processing for FTMS.• Advanced capabilities and powerful algorithms, but simple to use.• Support for single mass spectra and also FTMS imaging.• Support for data formats of common FTMS instruments and software.• Configured for petroleomic, proteomic or any high mass accuracy application.• Is compatible with Peak-by-Peak for easy integration and data co-processing.• Developed in collaboration with Dr. David Kilgour – www.kilgourlab.com.

See the original implementation for LTQ FT :

Producing absorption mode FT-ICR mass spectra with non-quadratic phase correction functions. Rapid Commun. Mass Spectrom., 2015, 29, 1087-1093

• A graphical user interface and built-in help system guide users through FTMS aFT data processing.

• Built-in tools include advanced peak detection (Autopiquer) and top-down protein sequencing (Autoseequer).

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CS(compressed sensing)

xFT(extended basis FT)

LSF(least-squares fitting)

FDM(filter diagonalization method)

ФSDM(phased spectrum deconvolution method)

mFT(magnitude mode FT)

eFT(enhanced FT = mFT + aFT)

aFT(absorption mode FT)

Fourier transform

Super-re

solu

tion

RESOLU

TION

Algorithms for FTMS transient processing supported by Peak-by-Peak :• mFT (magnitude mode FT): basic screening of the FTMS data, multicore processing.• aFT (absorption mode FT): doubles the mFT resolving power ; processing of both phased and unphased transients

from any FTMS instrument ; aFT resolution performance matches that of eFT (enhanced Fourier transform).• LSF (least-squares fitting): targeted identification of ion abundance for ions with a-priori known m / z values ; improved

resolving power, sensitivity, mass and abundance accuracy compared to FT methods. Implementation : Aushev et al., JASMS 2014, 1263-1273.

• FDM (filter-diagonalization method): untargeted reconstruction of mass spectra with similar advantages as with the LSF. Implementation : Kozhinov et al., Anal. Chem 2012, 2850-2856.

• xFT (extended basis FT): improves resolution for mass spectra with high harmonics content ; removes unwanted harmonics from FTMS mass spectra.

Peak-by-Peak : Advanced FTMS Data ProcessingMost FTMS instruments provide only processed and noise-reduced mass spectra. Spectroswiss gives access to FTMS transients, which allows for much more efficient and powerful data processing, including Fourier transform and super-resolution algorithms.

The accurately phased transients uniquely provided by FTMS Booster improve performance of all signal processing algorithms. In particular, they enable pure absorption mode FT spectra from any FTMS instrument, including OrbitrapTM FTMS and FT-ICR MS.

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Peak-by-Peak : Advanced FTMSPowerful and flexible software for advanced processing of FTMS dataTailored workflows for petroleomics, imaging, lipidomics, top-down, spectroscopy

Key features of Peak-by-Peak data processing :• Integration and processing of practically any size .RAW files, .d folders and .HDF5 files.• Open file formats for transients & mass spectra : HDF5, MGF, mzXML, mzML, imzXML.• Visualization of transients and mass spectra, visual comparison from separate data sets.• Spectral and transient summation for high sensitivity and dynamic range : data averaging across very long data sets,

e.g., 100,000 scans.• Basic discrete FT with parallel, multi-CPU computing.• Advanced FT with pre- and post-processing, including apodization, zero-filling, magnitude and absorption mode FT.• Noise thresholding, reduced-profile mode.• Peak picking with N-point interpolation and efficient baseline correction.• Mass calibration equations : (i) custom and standard calibration laws, (ii) pre-sets of calibration equations for FT-

ICRs with given magnetic field strengths and orbitrap mass analyzers of given geometry and trapping voltage ; (iii) iterative re-calibration procedure for complex mixture analysis (petroleomics).

• GPU-driven least-squares fitting (LSF) for real-life applications.• Handling of (very) large amounts of data, including imaging.• Parallel computing on multiple CPU-cores for fast data processing.• Cross-platform code supporting Windows and Linux.• Rapid development of custom software applications by the user and on-demand.

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Peak-by-Peak : LC MS / MSAdvanced mass spectra & transient processing improves LC MS / MS performance

Key features of Peak-by-Peak for LC-MS / MS data processing :• Visualization and processing of practically any size LC-MS / MS datasets (.RAW).• Workflows based on transients acquired with FTMS Booster.• Scan indexing between .RAW and transients datasets for every LC-MS / MS run.• Absorption mode FT of phased transients matches the performance of eFT processing.• Recalibration to improve mass accuracy.• Data summation across a number of LC-MS / MS runs, including chromatogram alignment.• Super-resolution signal processing.• Advanced control of processing parameters, including noise analysis.• Automated batch analysis of LC-MS / MS datasets.• Open file formats enable further analysis with commercial and open source tools.• Parallel computing on multiple CPU-cores for fast data processing.• Cross-platform code supporting Windows and Linux.

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Big Data : Petroleomics, ImagingProcessing of transients : conditional co-adding improves mass, abundance accuracy

Powerful re-calibration tools improve mass accuracy for petroleomics, imaging

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Validation and VizualizationMass accuracy plots for classes assigned using external software (PetroOrgTM)

Kendrick plots confirm data integrity for identified classes in petroleomics

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Petroleomics FT-ICR Data ProcessingData summation using transients compared to the reduced profile spectral summation improves sensitivity and provides up to 3-fold more analyte peaks per mass unit in a wider mass range, see below. Data acquisition was performed using FTMS Booster and data processing via Peak-by-Peak.

Analysis of mass spectra shown above. Left : number of analyte peaks per Da (300-1200 m / z) of a crude oil fraction with abundance higher than 6σnoise for (red) transient and (black) reduced profile spectral summation. Right : mass error distribution of 8784 monoisotopic peaks identified in transient summed absorption mode FT by AutoVectis.

Positive ion APPI FT-ICR MS (10 T LTQ FTTM, Thermo Scientific) of a crude oil fraction. Black : reduced profile spectral summation (.RAW, XcaliburTM). Red : transient summation (.H5, FTMS Booster). Both mass spectra are in magnitude mode FT and normalized to the same base peak. Preset resolution R=750,000 @ 400 m / z, AGC=off, IT=100 ms, 10 microscans per scan.

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Petroleomics FT-ICR Data ProcessingPeak-by-Peak and AutoVectis absorption mode FT compared to magnitude mode FT. Absorption mode provides similar resolution and improved sensitivity (more peaks detected) but the transient used is 3x shorter !

Application of iterative recalibration by Peak-by-Peak to the heavy oil sample mass spectrum shown above. The mass error distributions are (left) before and (right) after iterative recalibration. The procedure is described in : Kozhinov, Zhurov, and Tsybin ; Iterative method for mass spectra recalibration via empirical estimation of mass calibration function for FTMS-based petroleomics. Analytical Chemistry (2013) 85, 6437-6445

Positive ion ESI FT-ICR MS of a heavy oil sample. A summed transient containing 100 scans was acquired with a 12 T SolariXTM FT-ICR MS (Bruker Daltonics) and processed using Peak-by-Peak and AutoVectis. (Black) the transient with an original length of 3.3 s was processed in a magnitude mode and (Red) the transient was first truncated at 1 s and processed in absorption mode. The sensitivity increase is due to a substantial transient decay during the detection period.

before after

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All Ions All the Time : Full TransientsConventional FTMS data acquisition systems typically measure only a part of the available transient and sometimes provide mass spectra with a narrower m / z range than desired. FTMS Booster acquires full transients, maximizing the duty cycle, from all ions present in the mass analyzer.Full transient recording enables ion detection truly parallel to other processes, including accumulation and fragmentation (MS / MS) in external devices, such as electron transfer dissociation (ETD).Detecting all ions all the time improves the resolution, throughput, sensitivity, and the number of identified compounds.

Top-down MS performance on a QExactive HFTM OrbitrapTM FTMS is shown below. Peaks in black are present in the conventional .RAW mass spectrum. Peaks in red are present in the mass spectrum acquired and processed in parallel using FTMS Booster and Peak-by-Peak. Precursor protein size 20 kDa. HCD employed as MS/MS method. Precursor ion isolated at around 915.8 m/z.

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Proteomics on OrbitrapTM FTMSFTMS Booster and Peak-by-Peak software increase the productivity of OrbitrapTM FTMS in multiplexed protein quantitation with mass tags, including the N-plex TMTTM technology.

Example of an LC-MS / MS analysis of 10-plex TMTTM-labeled yeast digest on OrbitrapTM FusionTM LumosTM FTMS, with FTMS Booster and absorption mode FT (Peak-by-Peak, blue). Data in black show the OEM results (Thermo Scientific).

FTMS Booster acquires phased transients for absorption mode FT (aFT) mass spectra in parallel to mass spectra acquisition with OrbitrapTM FTMS in enhanced FT mode (eFT, .RAW). In case resolution provided by aFT is not sufficient, N-Plex Suite performs super-resolution (least-squares fitting, LSF) signal processing. Synergy of aFT and LSF signal processing allows for 2-4 fold increase in resolution compared to eFT, thus speeding up protein quantitation and allowing to quantify more peptides / proteins per LC MS / MS experiment. Both methods, aFT and LSF, demonstrate quantitatively-accurate performance even for very low SNR peaks. Thus, the number of missing values for protein quantitation, e.g., for TMTTM, can be reduced, benefiting clinical studies confidence.

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Multiplexed LC / GC-MS SensitivityGas chromatography / mass spectrometry (GC / MS) and liquid chromatography / mass spectrometry (LC / MS) are routinely used for proteomics and petroleomics applications. However, the sensitivity may be poor for low-abundant components due to a limited number of scans per elution peak. Conditional scan summation over a number of repetitions is an effective method for improving the measurement sensitivity. Multiplexed transient or spectral summation for a targeted elution peak is included in Peak-by-Peak software.

Multiplexed LC-MS / MS data processing application for top-down MS : Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer. Journal of Proteomics 2017, 67-76

Improving sensitivity by multiplexed summation (spectral or transient) across multiple chromatographic experiments (GC-MS or LC-MS). Data summed over a single run is compared to data summed over 10 runs.

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FTMS ImagingFTMS-based imaging greatly benefits from both hardware (FTMS Booster) and software (Peak-by-Peak and AutoVectis) solutions offered by Spectroswiss. Examples shown below demonstrate performance improvements for FT-ICR MS instruments from Thermo (top) and Bruker (bottom). The unique capabilities of Peak-by-Peak are essential for processing of large (5-10 GB) imaging datasets acquired with both ICR and OrbitrapTM FTMS instruments.

Positive ion MALDI imaging FT-ICR MS (9.4 T SolariXTM, Bruker Daltonics) of a rat brain. (Left) an expanded view into a mass spectrum generated from a single transient (.SER) represented in (black) magnitude mode FT and (red) absorption mode FT (AutoVectis) and normalized to the same base peak in a parent broadband mass spectrum. Resolution R=250’000 @ 800 m / z in the aFT mode. (Right) the corresponding images and the error (mass accuracy) distributions for the whole image (Peak-by-Peak).

Positive ion DESI imaging FT-ICR MS (7 T LTQ FTTM, Thermo Scientific) of a mouse kidney. Left : the reduced profile mass spectrum is in black. The corresponding mFT mass spectrum (acquired by FTMS Booster) is green, and red in aFT mode (AutoVectis). Pre-set resolution R=12,500 @ 400 m / z, AGC=off, IT=1000 ms which resulted in a boosted resolution R=250’000 @ 400 m/z. (Right) the corresponding images and the error (mass accuracy) distributions for the whole image (Peak-by-Peak).

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A word from our customers

Dr. Florian AlbrieuxFrench Institute of Petroleum (IFPEN), France

« We have been using Peak-by-Peak software since 2016 for processing and analysis of spectral and transient data acquired on our 7 T LTQ FT from petroleomic and ex-biomass samples. Peak-by-Peak enables summation of a large number of transients to provide an exceptional data quality, specifically in terms of a dynamic range and S / N, both in direct infusion and in chromatographic modes. In addition, it allows to perform conditional co-adding of transients based on frequency analysis, iterative re-calibration for improved mass accuracy, and provides flexible and tailored solutions as a function of a specific workflow employed. All in all, it dramatically increases the method efficiency for our petroleomics-type applications. »