Online coupling of a catalytic continuous microflow reactor to mass spectrometry

Flow cell reactors used for catalyst development and applications are upcoming due to their small environmental and economic footprint. Online microflow reactor coupling with mass spectrometry (MS) opens new possibilities for monitoring catalyst performance and identifying reaction products in real time. This is demonstrated for the metabolic relevant dealkylation of lidocaine on catalytic gold micro-particles using regular liquid chromatography modules. Yields of up to 90% norlidocaine were realized under mild continuous flow conditions for up to 10 h (pH 7, 30 °C and 20 μL/min). Dissolved oxygen was shown to be a rate-limiting factor, since an inline oxygen generator allowed to increase the reactor capacity by one order of magnitude. Monitoring product time-response curve slopes after starting and ending a substrate feed, provided insights into the adsorption/desorption and conversion kinetics at the catalyst surface indicating the presence of strong adsorption sites that do not contribute substantially to substrate conversion

Apex Peptide Elution Chain Selection: A New Strategy for Selecting Precursors in 2D-LC-MALDI-TOF/TOF Experiments on Complex Biological Samples

LC-MALDI provides an often overlooked opportunity to exploit the separation between LC-MS and MS/MS stages of a 2D-LC-MS-based proteomics experiment, that is, by making a smarter selection for precursor fragmentation. Apex Peptide Elution Chain Selection (APECS) is a simple and powerful method for intensity-based peptide selection in a complex sample separated by 2D-LC, using a MALDI-TOF/TOF instrument. It removes the peptide redundancy present in the adjacent first-dimension (typically strong cation exchange, SCX) fractions by constructing peptide elution profiles that link the precursor ions of the same peptide across SCX fractions. Subsequently, the precursor ion most likely to fragment successfully in a given profile is selected for fragmentation analysis, selecting on precursor intensity and absence of adjacent ions that may cofragment. To make the method independent of experiment-specific tolerance criteria, we introduce the concept of the branching factor, which measures the likelihood of false clustering of precursor ions based on past experiments. By validation with a complex proteome sample of Arabidopsis thaliana, APECS identified an equivalent number of peptides as a conventional data-dependent acquisition method but with a 35% smaller work load. Consequently, reduced sample depletion allowed further selection of lower signal-to-noise ratio precursor ions, leading to a larger number of identified unique peptides.

Online coupling of a catalytic continuous microflow reactor to mass spectrometry

Flow cell reactors used for catalyst development and applications are upcoming due to their small environmental and economic footprint. Online microflow reactor coupling with mass spectrometry (MS) opens new possibilities for monitoring catalyst performance and identifying reaction products in real time. This is demonstrated for the metabolic relevant dealkylation of lidocaine on catalytic gold micro-particles using regular liquid chromatography modules. Yields of up to 90% norlidocaine were realized under mild continuous flow conditions for up to 10 h (pH 7, 30 °C and 20 μL/min). Dissolved oxygen was shown to be a rate-limiting factor, since an inline oxygen generator allowed to increase the reactor capacity by one order of magnitude. Monitoring product time-response curve slopes after starting and ending a substrate feed, provided insights into the adsorption/desorption and conversion kinetics at the catalyst surface indicating the presence of strong adsorption sites that do not contribute substantially to substrate conversion

Online coupling of a catalytic continuous microflow reactor to mass spectrometry

Flow cell reactors used for catalyst development and applications are upcoming due to their small environmental and economic footprint. Online microflow reactor coupling with mass spectrometry (MS) opens new possibilities for monitoring catalyst performance and identifying reaction products in real time. This is demonstrated for the metabolic relevant dealkylation of lidocaine on catalytic gold micro-particles using regular liquid chromatography modules. Yields of up to 90% norlidocaine were realized under mild continuous flow conditions for up to 10 h (pH 7, 30 °C and 20 μL/min). Dissolved oxygen was shown to be a rate-limiting factor, since an inline oxygen generator allowed to increase the reactor capacity by one order of magnitude. Monitoring product time-response curve slopes after starting and ending a substrate feed, provided insights into the adsorption/desorption and conversion kinetics at the catalyst surface indicating the presence of strong adsorption sites that do not contribute substantially to substrate conversion

Online coupling of a catalytic continuous microflow reactor to mass spectrometry

Flow cell reactors used for catalyst development and applications are upcoming due to their small environmental and economic footprint. Online microflow reactor coupling with mass spectrometry (MS) opens new possibilities for monitoring catalyst performance and identifying reaction products in real time. This is demonstrated for the metabolic relevant dealkylation of lidocaine on catalytic gold micro-particles using regular liquid chromatography modules. Yields of up to 90% norlidocaine were realized under mild continuous flow conditions for up to 10 h (pH 7, 30 °C and 20 μL/min). Dissolved oxygen was shown to be a rate-limiting factor, since an inline oxygen generator allowed to increase the reactor capacity by one order of magnitude. Monitoring product time-response curve slopes after starting and ending a substrate feed, provided insights into the adsorption/desorption and conversion kinetics at the catalyst surface indicating the presence of strong adsorption sites that do not contribute substantially to substrate conversion

Online coupling of a catalytic continuous microflow reactor to mass spectrometry

Flow cell reactors used for catalyst development and applications are upcoming due to their small environmental and economic footprint. Online microflow reactor coupling with mass spectrometry (MS) opens new possibilities for monitoring catalyst performance and identifying reaction products in real time. This is demonstrated for the metabolic relevant dealkylation of lidocaine on catalytic gold micro-particles using regular liquid chromatography modules. Yields of up to 90% norlidocaine were realized under mild continuous flow conditions for up to 10 h (pH 7, 30 °C and 20 μL/min). Dissolved oxygen was shown to be a rate-limiting factor, since an inline oxygen generator allowed to increase the reactor capacity by one order of magnitude. Monitoring product time-response curve slopes after starting and ending a substrate feed, provided insights into the adsorption/desorption and conversion kinetics at the catalyst surface indicating the presence of strong adsorption sites that do not contribute substantially to substrate conversion

Online coupling of a catalytic continuous microflow reactor to mass spectrometry

Flow cell reactors used for catalyst development and applications are upcoming due to their small environmental and economic footprint. Online microflow reactor coupling with mass spectrometry (MS) opens new possibilities for monitoring catalyst performance and identifying reaction products in real time. This is demonstrated for the metabolic relevant dealkylation of lidocaine on catalytic gold micro-particles using regular liquid chromatography modules. Yields of up to 90% norlidocaine were realized under mild continuous flow conditions for up to 10 h (pH 7, 30 °C and 20 μL/min). Dissolved oxygen was shown to be a rate-limiting factor, since an inline oxygen generator allowed to increase the reactor capacity by one order of magnitude. Monitoring product time-response curve slopes after starting and ending a substrate feed, provided insights into the adsorption/desorption and conversion kinetics at the catalyst surface indicating the presence of strong adsorption sites that do not contribute substantially to substrate conversion

The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode

[Image: see text] Data-independent acquisition (DIA) is an increasingly used approach for quantitative proteomics. However, most current isotope labeling strategies are not suitable for DIA as they lead to more complex MS2 spectra or severe ratio distortion. As a result, DIA suffers from a lower throughput than data-dependent acquisition (DDA) due to a lower level of multiplexing. Herein, we synthesized an isotopically labeled acetyl-isoleucine-proline (Ac-IP) tag for multiplexed quantification in DIA. Differentially labeled peptides have distinct precursor ions carrying the quantitative information but identical MS2 spectra since the isotopically labeled Ac-Ile part leaves as a neutral loss upon collision-induced dissociation, while fragmentation of the peptide backbone generates regular fragment ions for identification. The Ac-IP-labeled samples can be analyzed using general DIA liquid chromatography–mass spectrometry settings, and the data obtained can be processed with established approaches. Relative quantification requires deconvolution of the isotope envelope of the respective precursor ions. Suitability of the Ac-IP tag is demonstrated with a triplex-labeled yeast proteome spiked with bovine serum albumin that was mixed at 10:5:1 ratios, resulting in measured ratios of 9.7:5.3:1.1

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes

Quantifying proteins based on peptide-coupled reporter ions is a multiplexed quantitative strategy in proteomics that alleviates the problem of ratio distortion caused by peptide cofragmentation, as commonly observed in other reporter-ion-based approaches, such as TMT and iTRAQ. Data-independent acquisition (DIA) is an attractive alternative to data-dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra, severe ratio distortion, or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag that conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter ions. Since the peptide-coupled reporter ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are identical, the Ac-AG tag is compatible with both DDA and DIA. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter ions represent the relative ratios between constituent samples, whereas in DIA, the ratio can be inferred after deconvoluting the peptide-coupled reporter ion isotopes. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within this complex proteomics background, BSA spiked at 1:5:10 ratios was detected at ratios of 1.00:4.87:10.13 in DDA and 1.16:5.20:9.64 in DIA

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitating data processing. Multiplex labeling is based on selective N-terminal dimethylation followed by derivatization of the ε-amino group of the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags having complementary isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y ions, with the neutral loss of Ac-Ile, can be distinguished between the different labeling channels based on different numbers of isotope labels on the Pro-Gly part and thus contain the information for relative quantification, while b ions of different labeling channels have the same m/z values. The proteome quantification capability of this method was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. With the yeast proteins as the background, BSA was detected at ratios of 1.14:5.06:9.78 when spiked at 1:5:10 ratios. The raw mass data is available on the ProteomeXchange with the identifier PXD 018790