Application Note # LCMS-62 Walk-Up Ion Trap Mass Spectrometer System in a Multi-User Environment Using Compass OpenAccess Software

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1 Application Note # LCMS-62 Walk-Up Ion Trap Mass Spectrometer System in a Multi-User Environment Using Compass OpenAccess Software Abstract Presented here is a case study of a walk-up liquid chromatography - ion trap mass spectrometry (LC-MS) system at the School of Chemistry at the University of Leeds. The system allows for walk-up access to a Bruker HCTultra LC-MS system. The implementation of client-server based Compass OpenAccess software on this system allows the user to choose from several pre-programmed LC-MS workflows with no need for tedious method development or system optimisation (Figure 1). Current workflows span from small molecule quality control (QC) to protein identification. User training is minimal as is system down time. The system at the University of Leeds currently has over 100 users and runs approximately 20,000 samples a year. Introduction Mass spectrometric analysis (MS) has been routine in the characterisation of molecules for many years and the analytical power of the technology is commonly increased by the introduction of a high performance liquid chromatography (HPLC) front end. The development of rapid chromatographic methods along with robust ionisation techniques means that the timescale for LC-MS analysis is frequently only minutes making the tool ideal for real time monitoring of reaction progress and rapid quality control (QC) work. However, access to analytical instrumentation is frequently limited by the training required to enable users to successfully operate both LC and MS instrumentation. In some cases improper instrument use can result in significant laboratory down-time. The Bruker Compass OpenAccess system is a multi-user software tool that eliminates this learning curve allowing users new to LC-MS the ability to generate valuable data with a minimal amount of training. Here, we outline how this technology is enabling the students, technicians and researchers at the University of Leeds the opportunity to solve analytical problems using LC-MS with minimal instrument training. The system consists of an HPLC with an autosampler and column switching valve coupled to a Bruker HCTultra ion trap MS. The added value of ion trap technology comes from the fact that ions can be isolated and fragmented once (MS/ MS) or several times (MS n ) to generate a large amount of compound specific data in a very short period of time. The Compass OpenAccess system at the University of Leeds currently has over 100 registered users and runs over 20,000 analyses a year. Here we provide the technical details of the system, examples of common workflows and user statistics.

2 System description The Compass OpenAccess LC-MS system at the University of Leeds consists of an Agilent 1200 binary HPLC, equipped with an autosampler, column temperature control compartment, a six port column switching valve and a UV/ Vis detector coupled to a Bruker HCTultra ion trap mass spectrometer equipped with a standard electrospray source (ESI). All of the latter LC-MS system modules are controlled through Compass HyStar software. User training and instrument maintenance At the University of Leeds, each user is required to watch a ten minute training video on the web prior to being registered. This video covers the essentials of sample submission and basic ion trap theory. Users are encouraged to practice mass spectrometry friendly sample preparation, but significant downtime due to sample contamination or carryover is nonexistent. System maintenance is also minimal due to the robustness of the Bruker HCTultra ion trap. Compass OpenAccess environment description Compass is Bruker s unified software environment for all mass spectrometry products. The Compass OpenAccess environment consists of a database installed on a central server that can be accessed anywhere via the web, along with a small piece of client software installed on the mass spectrometer acquisition PC that manages the link between Compass OpenAccess and the Compass HyStar control systems (Figure 1). The administrative team at Leeds have created a range of generic LC-MS methods suitable for different applications which the user can select from at the point of sample submission and which provide good analytical conditions for the vast majority of projects. More complex or custom methods and workflows can easily be added by the administrator. The usage of the system speaks for itself. The fact that a range of chemists from across the breadth of the discipline are constantly submitting samples to the instrument 7 days a week shows the value that the research chemists place in having rapid turnaround access to high quality LC-MS data Dr. Stuart Warriner Client server configuration Figure 1. A client-server configuration allows modular growth of the Compass OpenAccess platform and decentralised web-based data access with multiple instrument configurations.

3 Sample submission When a user is ready to run a sample, the web-based Compass OpenAccess system is accessed and the sample data is submitted. The submission process is intuitively obvious and the web-based system requires no special software installation. Samples can be submitted either one at a time or in a sequence depending on the needs of the user (Figure 2). The Compass OpenAccess user then inputs a sample name, a description and chooses one of the predefined LC-MS methods (Figure 3). Large sample lists can also be uploaded from an excel spreadsheet. Once a method is chosen, the system asks for a few details which are specific to that method, such as target molecular formulas (Figure 4). When the user is ready to physically submit their sample, a simple dialogue indicates the next available rack position (Figure 5). The sample is placed in the requested location and enters the sample queue. Upon method completion, the user can receive a pdf result report of the processed LC-MS analysis either by logging back into the system or via . Users also have the option of requesting a hard copy printout. The simple client interface provides instrument status as well as letting users know the length of the sample queue (Figure 6). Compass OpenAccess workflow Figure 2: Web-based Compass OpenAccess sample submission. The server is accessed from any computer with an internet connection. Figure 3: Example of a sample submission. The user inputs a sample name, a description and then chooses the specific LC-MS method required. Figure 6: Compass OpenAccess client displaying the instrument status. The actual instrument control software is invisible to the user. Figure 5: Compass OpenAccess tells the user where to place their sample in the autosampler. Once the sample is placed, the user checks the required box and the sample is queued. Figure 4: Depending on the method, additional sample data can be entered by the user, in this case, the expected chemical formulas of the starting material and the product along with a request to print a hard copy of the report.

4 Workflow examples Much of the power of the Compass OpenAccess system comes from the highly customisable workflows which can be easily configured by the site administrator. The system can be customised to have as many or as few pre-defined LC-MS workflows as needed. Each workflow consists of the required LC, MS and data-processing steps. The dataprocessing steps can utilise all the data provided by the user at sample submission making it easy to create output reports which optimise the presentation of data to the user. QC for chemical synthesis The most common use for the LC-MS system at Leeds is for monitoring reactions and assessing if the reaction has reached completion and/or if the correct product has been formed. The user simply submits their sample, optionally specifying up to two masses or formulas of interest. The output allows the user to identify compounds that have been formed. If mass/formula targets have been specified then extracted ion chromatograms are generated for these masses which can aid data interpretation. Mass spectra are automatically extracted for the most significant peaks (see Figure 7). LC-MS Report Quality control of proteins The Compass OpenAccess system has also proved to be especially useful for the chemical biologists working at Leeds. Several projects require the chemical derivatisation of expressed proteins with low molecular weight tags. Careful control of the reactions is assisted by the ability to monitor derivatisation in real time using Compass Open Access. The samples can either be run in LC-MS mode or without chromatographic separation depending on the sample format and user requirements. The charge state envelope is automatically deconvoluted to calculate the molecular weight of the proteins (Figure 8). The robust nature of the HCTultra ion trap and LC-system means that no set up changes are required for protein work and the Compass OpenAccess system continuously provides both biological and small molecule analytical support without any external intervention. Protein Report A B C Figure 7. A typical LC-MS report showing MS and UV chromatograms, extracted ion chromatograms and mass spectra for key peaks. The inset shows the mass spectra of both the starting material and the product. Figure 8. Compass OpenAccess acquired full scan MS spectra of proteins. The top panel shows the derivatisation of Chorlera toxin B (CTB). Mass spectra of (A) CTB monomer and (B) derivatised CTB. The insets show how the deconvolution routine easily shows the small net mass change following oxidation and oxime formation (C). The system also produces excellent quality spectra of larger protein such as maltose binding protein. Data courtesy of T. Branson and Dr. W.B. Turnbull.

5 MS/MS analysis The Leeds OpenAccess system is equipped with an HCTultra ion trap which enables the power of MS/MS and MS n experiments to also be accessed. LC-MS/MS experiments provide automated fragmentation of the most intense ion(s) at any time to augment the identification of components in reaction mixtures. LC separation is not always used within the workflows. The system is configured with a bypass loop to give users the option just to obtain a simple analysis of compounds in either positive or negative ionisation mode without liquid chromatography separation. In this example, the method automatically acquires a full scan spectrum and an MS/MS spectrum of the most intense precursor ion from the sample (Figure 9). Walk up acquisition including generation of the shown report only takes one minute. Data analysis In addition to pdf reports, a web-based viewer allows the user to analyse the processed data of more complex samples in detail (Figure 10). The Leeds setup also enables the user to reprocess their data using the full power of Compass DataAnalysis software at a dedicated workstation. Advanced administrative tools Compass OpenAccess also provides the system administrator with a range of tools that make day-to-day system management as easy as possible. Creation of users is easily performed either on an ad-hoc basis through a web interface or in batches from an uploaded excel file. Users are associated with supervisors and departments and this structure is also mapped onto the data storage architectures making it very easy to manage large quantities of data. Automated backups of the Compass OpenAccess database also assist in ensuring data safety. The management tools also include simple methods to contact all users by as well as providing alerts to the system administrator in the rare event of an error. The database architecture also makes it easy to extract significant user, sample and instrument statistics for accounting purposes (Figure 11). System usage statistics are also continuously monitored providing the administrator information about when the instrument is being used by hour, day, month or year (Figure 12). MS/MS analysis Standalone viewer A B Figure 9. Walk up MS-MS Analysis results. The system automatically acquires a full scan MS spectrum (A) and then selects the most intense ion for MS-MS acquisition (B). Data courtesy of M. Dow and Prof A. Nelson. Figure 10. Results can be reviewed in the web-based standalone viewer remotely.

6 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics , LCMS-62, # Conclusions The Bruker Compass OpenAccess software provides a very useful environment to allow multiple users with little to no training to generate invaluable MS, LC-MS and even LC-MS/MS data to help them answer their analytical questions. This platform is a versatile and beneficial tool for the introduction of LC-MS into any laboratory. Compass OpenAccess workflow There is no doubt that hands-on, walk-up access to high quality LC-MS is changing the way we are doing science at Leeds. Not only are we benefiting from improved productivity from our researchers but, increasingly, we are also tackling scientific problems in ways that simply would not be possible without this kind of rapid turnaround analytical support. Dr. Stuart Warriner Figure 11: User Accounting System usage Keywords walk up open access multi-user Instrumentation & Software Ion trap Compass OpenAccess Authors Stuart Warriner, Senior Lecturer, School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK P. John Wright, Bruker Daltonics, 3500 Warren Ave., Fremont, CA 94538, USA Sebastian Goetz, Bruker Daltonik GmbH, Fahrenheitstraße 4, Bremen, Germany For research use only. Not for use in diagnostic procedures. Figure 12. The open access nature of the systems enables it to support key experiments that demand data outside the normal working day. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Fax +49 (0) Bruker Daltonics Inc. Billerica, MA USA Fremont, CA USA Phone +1 (978) Phone +1 (510) Fax +1 (978) Fax +1 (510)

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