Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.     

Analysis of oxysterol metabolomes

Oxysterols are oxygenated forms of cholesterol. This definition can, however, be expanded to include oxygenated derivatives of plant sterols and also of cholesterol precursors. Oxysterols are formed in the first steps of cholesterol metabolism and also from cholesterol by reactive oxygen species. Oxysterols were once thought of as simple intermediates, or side-products, in the conversion of cholesterol to hormonal steroids and bile acids, however, they have subsequently been shown to be biologically active molecules in their own right. In this article we will discuss methods of oxysterol analysis including "classical" gas chromatography-mass spectrometry (GC-MS) methods and more recent liquid chromatography (LC)-MS methods. Our main focus, however, will be on analytical methods based on "charge-tagging" and LC-tandem mass spectrometry (MS/MS or MS(n)) which we have developed over the last decade in our laboratory. Examples will be given of oxysterol analysis in brain, cerebrospinal fluid (CSF) and blood. The advantages and disadvantages of the various methods of oxysterol analysis will be discussed.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.     

Quantitative secondary ion monitoring gas chromatography/mass spectrometry of diethylstilbestrol in bovine liver

A procedure is described for the extraction of diethylstilbestrol (DES) from animal tissue for quantitative capillary gas chromatography/mass spectrometry (GC/MS). The procedure is based upon use of a strong anion exchange polystyrene divinylbenzene resin for sample purification. The recovery of DES from the resin clean up was 88% in the high parts per trillion (ppt) range. Criteria for identification of DES using selected ion monitoring (SIM) GC/MS are presented. Liquid chromatography/mass spectrometry (LC/MS) was used to investigate altered DES cis/trans ratios observed in biological extracts.     

Analysis of human antibody IgG2 domains by reversed-phase liquid chromatography and mass spectrometry

It has been well documented that papain cleaves an IgG1 molecule to release Fab and Fc domains; however, papain was found unable to release such domains from an IgG2. Here we present a new combinatory strategy to analyze the heterogeneity of the light chain (LC), single chain Fc (sFc), and Fab portion of the heavy chain (Fd) of an IgG2 molecule released by papain cleavage under mild reducing conditions. These domains were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by in-line liquid chromatography time-of-flight mass spectrometry (LC–TOF/MS). In addition, some modifications of these domains were revealed by in-line mass spectrometry, and confirmed by the peptide mapping on LC–MS/MS analysis. This same strategy was proven suitable for IgG1 molecules as well. This procedure provides a simplified approach for the characterization of antibody biomolecules by facilitating the detection of low-level modifications in a domain. In addition, the technique offers a new strategy as an identification assay to distinguish IgG2 molecules on RP-HPLC, by which highly conserved Fc domains remain at a constant retention time (RT) unique to its subisotype, while varying RTs of the light chain and the Fd distinguish the monoclonal antibody from other molecules of the same isotype based on the underlying characteristics of each antibody.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.     

Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry

Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins 相似文献   

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10⁶ DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10⁶ DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Advanced mass spectrometric methods for the rapid and quantitative characterization of proteomes

Progress is reviewed towards the development of a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of high performance separations and mass spectrometry. The approach uses high accuracy mass measurements from Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate peptide 'accurate mass tags' (AMTs) produced by global protein enzymatic digestions for a specific organism, tissue or cell type from 'potential mass tags' tentatively identified using conventional tandem mass spectrometry (MS/MS). This provides the basis for subsequent measurements without the need for MS/ MS. High resolution capillary liquid chromatography separations combined with high sensitivity, and high resolution accurate FTICR measurements are shown to be capable of characterizing peptide mixtures of more than 10(5) components. The strategy has been initially demonstrated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, high sensitivity, and the capability for stableisotope labeling methods for precise relative protein abundance measurements.Abbreviations: LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; AMT, accurate mass tag; PMT, potential mass tag; MMA, mass measurement accuracy; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ppm, parts per million.     

Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data

Liquid chromatography–mass spectrometry (LC–MS) is a commonly used analytical platform for non-targeted metabolite profiling experiments. Although data acquisition, processing and statistical analyses are almost routine in such experiments, further annotation and subsequent identification of chemical compounds are not. For identification, tandem mass spectra provide valuable information towards the structure of chemical compounds. These are typically acquired online, in data-dependent mode, or offline, using handcrafted acquisition methods and manually extracted from raw data. Here, we present several methods to fast-track and improve both the acquisition and processing of LC–MS/MS data. Our nearly online (nearline) data-dependent tandem MS strategy creates a minimal set of LC–MS/MS acquisition methods for relevant features revealed by a preceding non-targeted profiling experiment. Using different filtering criteria, such as intensity or ion type, the acquisition of irrelevant spectra is minimized. Afterwards, LC–MS/MS raw data are processed with feature detection and grouping algorithms. The extracted tandem mass spectra can be used for both library search and de-novo identification methods. The algorithms are implemented in the R package MetShot and support the export to Bruker, Agilent or Waters QTOF instruments and the vendor-independent TraML standard. We evaluate the performance of our workflow on a Bruker micrOTOF-Q by comparison of automatically acquired and extracted tandem mass spectra obtained from a mixture of natural product standards against manually extracted reference spectra. Using Arabidopsis thaliana wild-type and biosynthetic gene knockout plants, we characterize the metabolic products of a biosynthetic pathway and demonstrate the integration of our approach into a typical non-targeted metabolite profiling workflow.