This talk focuses on high-resolution accurate mass (HR/AM) analysis using Orbitrap mass spectrometry. Basic principles of this technology are presented against a backdrop of its brief but colourful historic development. Since its commercial launch 15 years ago, the utility of the Orbitrap analyser has been continuously extended by coupling with additional capabilities such as quantitative analysis, new fragmentation methods, different vacuum and ambient ion sources, imaging and ion mobility. These capabilities are exemplified for four major families of Orbitrap-based instruments, with numerous new modes of operation enabled by parallelization of detection and ion processing, and intricate coordination with different ion-optical devices. New modes of data-independent, targeted and top-down analysis are overviewed, including acquisitions at 40 spectra/second or at 1 million resolution setting. The roadmap of future instrument innovations is overviewed. Major directions of progress include trap and instrument designs, analytical modalities, acquisition methods as well as signal processing. Special attention is devoted to analysis of proteins and protein complexes that poses unique challenges to mass spectrometry and drives the deep re-thinking of principles earlier validated on small molecules and peptides. In conclusion, future trends and perspectives of Orbitrap mass spectrometry are discussed, including its inroads into emerging areas of mass spectrometric analysis. It is shown that Orbitrap-based mass spectrometers possess compelling potential as an (ultra-) high resolution platform not only for high-end proteomic applications but also for screening, trace and targeted analysis by LC/ and GC/MS.

This talk focuses on high-resolution accurate mass (HR/AM) analysis using Orbitrap mass spectrometry. Basic principles of this technology are presented against a backdrop of its brief but colourful historic development. Since its commercial launch 15 years ago, the utility of the Orbitrap analyser has been continuously extended by coupling with additional capabilities such as quantitative analysis, new fragmentation methods, different vacuum and ambient ion sources, imaging and ion mobility. These capabilities are exemplified for four major families of Orbitrap-based instruments, with numerous new modes of operation enabled by parallelization of detection and ion processing, and intricate coordination with different ion-optical devices. New modes of data-independent, targeted and top-down analysis are overviewed, including acquisitions at 40 spectra/second or at 1 million resolution setting. The roadmap of future instrument innovations is overviewed. Major directions of progress include trap and instrument designs, analytical modalities, acquisition methods as well as signal processing. Special attention is devoted to analysis of proteins and protein complexes that poses unique challenges to mass spectrometry and drives the deep re-thinking of principles earlier validated on small molecules and peptides. In conclusion, future trends and perspectives of Orbitrap mass spectrometry are discussed, including its inroads into emerging areas of mass spectrometric analysis. It is shown that Orbitrap-based mass spectrometers possess compelling potential as an (ultra-) high resolution platform not only for high-end proteomic applications but also for screening, trace and targeted analysis by LC/ and GC/MS.

This talk focuses on high-resolution accurate mass (HR/AM) analysis using Orbitrap mass spectrometry. Basic principles of this technology are presented against a backdrop of its brief but colourful historic development. Since its commercial launch 15 years ago, the utility of the Orbitrap analyser has been continuously extended by coupling with additional capabilities such as quantitative analysis, new fragmentation methods, different vacuum and ambient ion sources, imaging and ion mobility. These capabilities are exemplified for four major families of Orbitrap-based instruments, with numerous new modes of operation enabled by parallelization of detection and ion processing, and intricate coordination with different ion-optical devices. New modes of data-independent, targeted and top-down analysis are overviewed, including acquisitions at 40 spectra/second or at 1 million resolution setting. The roadmap of future instrument innovations is overviewed. Major directions of progress include trap and instrument designs, analytical modalities, acquisition methods as well as signal processing. Special attention is devoted to analysis of proteins and protein complexes that poses unique challenges to mass spectrometry and drives the deep re-thinking of principles earlier validated on small molecules and peptides. In conclusion, future trends and perspectives of Orbitrap mass spectrometry are discussed, including its inroads into emerging areas of mass spectrometric analysis. It is shown that Orbitrap-based mass spectrometers possess compelling potential as an (ultra-) high resolution platform not only for high-end proteomic applications but also for screening, trace and targeted analysis by LC/ and GC/MS.

Mass spectrometry (MS) has become the primary choice to study the composition and dynamics of proteomes in a global and unbiased manner [1]. Starting from tedious workflows covering only a handful of proteins in the beginnings, contemporary workflows quantify thousands to ten thousand proteins in a wide range of biological samples with ever-increasing throughput. This development has been largely driven by technological advances, not at least the invention of the Orbitrap mass analyser 20 years ago [2]. Yet, many challenges remain toward the ultimate goal of ubiquitous and complete proteomes. For example, studies of large sample cohorts can suffer from limited robustness and sparsity of the resulting data matrices, in particular for lowabundance proteins. Data-independent acquisition holds great promise to alleviate such effects and is becoming an increasingly attractive strategy as the speed of Orbitrap mass analysis increases. Another challenge arises from the fact that proteins readily span ten orders of magnitude in abundance, and even more in extreme cases such as body fluids. To address this challenge, we have recently developed a method termed BoxCar that increases the dynamic range of Orbitrap MS1 scans by one order of magnitude [3] and we are currently exploring its benefits in various acquisition schemes. Increasing computational power now also allows analysing MS data in realtime [4], which will be the basis for smart data acquisition strategies in the future.

Mass spectrometry (MS) has become the primary choice to study the composition and dynamics of proteomes in a global and unbiased manner [1]. Starting from tedious workflows covering only a handful of proteins in the beginnings, contemporary workflows quantify thousands to ten thousand proteins in a wide range of biological samples with ever-increasing throughput. This development has been largely driven by technological advances, not at least the invention of the Orbitrap mass analyser 20 years ago [2]. Yet, many challenges remain toward the ultimate goal of ubiquitous and complete proteomes. For example, studies of large sample cohorts can suffer from limited robustness and sparsity of the resulting data matrices, in particular for lowabundance proteins. Data-independent acquisition holds great promise to alleviate such effects and is becoming an increasingly attractive strategy as the speed of Orbitrap mass analysis increases. Another challenge arises from the fact that proteins readily span ten orders of magnitude in abundance, and even more in extreme cases such as body fluids. To address this challenge, we have recently developed a method termed BoxCar that increases the dynamic range of Orbitrap MS1 scans by one order of magnitude [3] and we are currently exploring its benefits in various acquisition schemes. Increasing computational power now also allows analysing MS data in realtime [4], which will be the basis for smart data acquisition strategies in the future.

Mass spectrometry (MS) has become the primary choice to study the composition and dynamics of proteomes in a global and unbiased manner [1]. Starting from tedious workflows covering only a handful of proteins in the beginnings, contemporary workflows quantify thousands to ten thousand proteins in a wide range of biological samples with ever-increasing throughput. This development has been largely driven by technological advances, not at least the invention of the Orbitrap mass analyser 20 years ago [2]. Yet, many challenges remain toward the ultimate goal of ubiquitous and complete proteomes. For example, studies of large sample cohorts can suffer from limited robustness and sparsity of the resulting data matrices, in particular for lowabundance proteins. Data-independent acquisition holds great promise to alleviate such effects and is becoming an increasingly attractive strategy as the speed of Orbitrap mass analysis increases. Another challenge arises from the fact that proteins readily span ten orders of magnitude in abundance, and even more in extreme cases such as body fluids. To address this challenge, we have recently developed a method termed BoxCar that increases the dynamic range of Orbitrap MS1 scans by one order of magnitude [3] and we are currently exploring its benefits in various acquisition schemes. Increasing computational power now also allows analysing MS data in realtime [4], which will be the basis for smart data acquisition strategies in the future.

With every year, Orbitrap FTMS technology is employed to address increasingly challenging applications. To support this growth, the outmost care is to be given to all experimental aspects. Acquisition of time-domain ion signals (transients) and their appropriate processing to yield artefact-free mass spectra are among the key aspects of the Orbitrap FTMS measurement process. Synergistic development of newgeneration architectures for data acquisition systems and mathematical algorithms for signal processing and big data handling greatly contributes to the opportunities offered by Orbitrap FTMS enabling novel capabilities and workflows. Previously, we implemented a high-performance data acquisition technology enabling users to access transients from FTMS instruments (ICR or Orbitrap) in parallel with the regular mass spectra acquisition. The flexibility of parallel data acquisition approach allowed us to maximize the duty cycle of ion detection, enabling acquisition of extended length transients. These developments, together with the progress in allied data processing algorithms of both transients and mass spectra, expanded user-controlled capabilities for Orbitrap FTMS experiment and maximized its value for applications. For example, we demonstrated the benefits of inter-experiment averaging of transients, as well as full and reduced profile mass spectra, for top-down and middle-down Orbitrap FTMS of monoclonal antibodies and other glycoproteins [1, 2]. Recently, we extended this approach to enable inter-experiment averaging of transients from technical replicates in GC Orbitrap FTMS to enhance its sensitivity and quantitation accuracy performance for trace-level analysis of persistent organic pollutants [3]. In this presentation, we will first describe some of the best practices of data acquisition and data processing for Orbitrap FTMS. In the following we will discuss the value of advanced data acquisition and processing approaches through selected examples of Orbitrap FTMS performance improvement and related application benefits.

With every year, Orbitrap FTMS technology is employed to address increasingly challenging applications. To support this growth, the outmost care is to be given to all experimental aspects. Acquisition of time-domain ion signals (transients) and their appropriate processing to yield artefact-free mass spectra are among the key aspects of the Orbitrap FTMS measurement process. Synergistic development of newgeneration architectures for data acquisition systems and mathematical algorithms for signal processing and big data handling greatly contributes to the opportunities offered by Orbitrap FTMS enabling novel capabilities and workflows. Previously, we implemented a high-performance data acquisition technology enabling users to access transients from FTMS instruments (ICR or Orbitrap) in parallel with the regular mass spectra acquisition. The flexibility of parallel data acquisition approach allowed us to maximize the duty cycle of ion detection, enabling acquisition of extended length transients. These developments, together with the progress in allied data processing algorithms of both transients and mass spectra, expanded user-controlled capabilities for Orbitrap FTMS experiment and maximized its value for applications. For example, we demonstrated the benefits of inter-experiment averaging of transients, as well as full and reduced profile mass spectra, for top-down and middle-down Orbitrap FTMS of monoclonal antibodies and other glycoproteins [1, 2]. Recently, we extended this approach to enable inter-experiment averaging of transients from technical replicates in GC Orbitrap FTMS to enhance its sensitivity and quantitation accuracy performance for trace-level analysis of persistent organic pollutants [3]. In this presentation, we will first describe some of the best practices of data acquisition and data processing for Orbitrap FTMS. In the following we will discuss the value of advanced data acquisition and processing approaches through selected examples of Orbitrap FTMS performance improvement and related application benefits.

With every year, Orbitrap FTMS technology is employed to address increasingly challenging applications. To support this growth, the outmost care is to be given to all experimental aspects. Acquisition of time-domain ion signals (transients) and their appropriate processing to yield artefact-free mass spectra are among the key aspects of the Orbitrap FTMS measurement process. Synergistic development of newgeneration architectures for data acquisition systems and mathematical algorithms for signal processing and big data handling greatly contributes to the opportunities offered by Orbitrap FTMS enabling novel capabilities and workflows. Previously, we implemented a high-performance data acquisition technology enabling users to access transients from FTMS instruments (ICR or Orbitrap) in parallel with the regular mass spectra acquisition. The flexibility of parallel data acquisition approach allowed us to maximize the duty cycle of ion detection, enabling acquisition of extended length transients. These developments, together with the progress in allied data processing algorithms of both transients and mass spectra, expanded user-controlled capabilities for Orbitrap FTMS experiment and maximized its value for applications. For example, we demonstrated the benefits of inter-experiment averaging of transients, as well as full and reduced profile mass spectra, for top-down and middle-down Orbitrap FTMS of monoclonal antibodies and other glycoproteins [1, 2]. Recently, we extended this approach to enable inter-experiment averaging of transients from technical replicates in GC Orbitrap FTMS to enhance its sensitivity and quantitation accuracy performance for trace-level analysis of persistent organic pollutants [3]. In this presentation, we will first describe some of the best practices of data acquisition and data processing for Orbitrap FTMS. In the following we will discuss the value of advanced data acquisition and processing approaches through selected examples of Orbitrap FTMS performance improvement and related application benefits.

Proteomics allows the identification and quantification of thousands of proteins in a sample. This capability designates proteomics as a preferred technique for the analysis of clinical samples. However, the low throughput of samples hampers the widespread application of proteomics with a significant analysis depth. The use of new chromatographic setups in combination with high-resolution mass spectrometry enables the analysis of proteomics samples in ultra-short gradients. Here the use of very high-resolution scans can be utilized for the removal of interfering signals and allowing for a deeper probing of the proteome with increased identification robustness across all samples.

Proteomics allows the identification and quantification of thousands of proteins in a sample. This capability designates proteomics as a preferred technique for the analysis of clinical samples. However, the low throughput of samples hampers the widespread application of proteomics with a significant analysis depth. The use of new chromatographic setups in combination with high-resolution mass spectrometry enables the analysis of proteomics samples in ultra-short gradients. Here the use of very high-resolution scans can be utilized for the removal of interfering signals and allowing for a deeper probing of the proteome with increased identification robustness across all samples.

Proteomics allows the identification and quantification of thousands of proteins in a sample. This capability designates proteomics as a preferred technique for the analysis of clinical samples. However, the low throughput of samples hampers the widespread application of proteomics with a significant analysis depth. The use of new chromatographic setups in combination with high-resolution mass spectrometry enables the analysis of proteomics samples in ultra-short gradients. Here the use of very high-resolution scans can be utilized for the removal of interfering signals and allowing for a deeper probing of the proteome with increased identification robustness across all samples.