Automation of MALDI-TOF Analysis for Proteomics

Transcription

1 Application Note #MT-50 BIFLEX III / REFLEX III Automation of MALDI-TOF Analysis for Proteomics D. Suckau, C. Köster, P.Hufnagel, K.-O. Kräuter and U. Rapp, Bruker Daltonik GmbH, Bremen, Germany Introduction Proteomics projects nowadays call for high throughput mass spectrometry. At present, the only technique which can cope with this demand is MALDI-TOF mass spectrometry. Bruker has introduced a high degree of automation on the MALDI-TOF mass spectrometers BIFLEX III and REFLEX III, resulting in MS analysis times below 1 minute per sample at excellent data quality. Automation has been achieved on different levels of analysis: MALDI sample preparation using a laboratory robot system (MAP II, MaldiAutoPrep) using 384 well-shaped MALDI targets Automatic spectra acquisition with real-time fuzzy logiccontrolled software (AutoXecute) Introduction of an algorithm (SNAP) for the fully automated monoisotopic peak annotations in the acquired spectra Performing automatic protein sequence library searches on the Internet as well as the Intranet under full control of the processing software. Preparation of samples Fig. 1 shows the scheme of proteomics sample preparations form the 2D gel containing the proteins to be investigated to the prepared samples on the MALDI target. Recently, excision robots became available, which excise stained protein spots from the gel as discs and deposit them in 96 well microtitreplates. The coding of the samples starts here and is related to the microplate nomenclature A1 to H12, in addition to the bar-coded well plate. Proteolytic digests are done in the plates. This step can be performed with several plates in parallel. 2D Gel Preparation for MALDI The plates with digests can be directly transferred to a liquid handling robot such as the Bruker MAP II (with 1 or 8 pipettes, Fig. 2), which prepares the samples for MALDI- TOF according to specific protocols, such as thin layer or dried droplet. Optional sample clean up can be selected, e.g. using Millipore ZipTips or on-target washing. The MAP II preparation methods can be defined in a Microsoft Excel based sample spreadsheet (Fig. 4). Such a spreadsheet also defines the automated acquisition and processing methods desired for these samples. Even robots with pipetting arrays for high throughput are compatible with the MALDI sample preparation, since the Bruker SCOUT 384 ion source uses sample targets fully compatible with the microtitreplate format (Fig. 3). Standard is the 384-sample target. Fig. 2: The MAP II robot for automatic MALDI sample preparation. Microtitreplate Microtitreplate 1. Excision robot 2. Digest MALDI sample plate Fig. 1: Preparation of proteomics samples from 2D gels Bruker Daltonik GmbH Fig. 3: The SCOUT 384 MALDI sample plate is 1:1 compatible with standard microtitreplates.

2 Robot pr ogr am MALDI Acquis it ion Fig. 4: The MAP Control spreadsheet controls automatic sample preparation, spectra acquisition and data analysis. Parameters covered are e.g. a sample ID, the position on the target, the preparation method and the analysis method. Automatic Acquisition Fig. 5 and 6 show spectra acquired automatically under fuzzy logic control using the Bruker AutoXecute software on a REFLEX III MALDI-TOF mass spectrometer. The fuzzy logic module judges the spectra quality "on the fly, optimizes the instrument's parameters and decides which spectra are to be summed up 1,2. 10 amol 100 amol Experience in several labs has shown that with this fuzzy logic-based acquisition the data quality increases dramatically and, as a consequence, so does the frequency of positive identifications during library searches. In contrast, simple algorithms without feed-back control of the laser fluence are incompatible with high quality data, especially in cases of small sample amounts or high sample complexity. The better the quality of peptide map spectra, the better and more reliable are the search results. Spectra can be acquired under automatic instrument control at high speed and at a quality comparable to manual control by an experienced instrument operator. Since the laser power is automatically adjusted to optimize spectra quality, variation of sample quality or concentration can be easily handled by the fuzzy logic control. 1 fmol Fig. 6: The real-time spectra judgement by a fuzzy logic module results in spectra of highest quality, as can be seen in this spectrum of a tryptic digest. 10 fmol Manual Fig. 5: Sensitivity is a crucial factor in proteome analysis. In the dilution series of the peptide VNKITVNKITVNKIG, even a sample amount of 10 attomole delivers clear and well-resolved spectra from automated analysis on the REFLEX III MALDI-TOF MS. Automatic Bruker Daltonik GmbH

3 Automatic Processing and Library Searches After data acquisition, the system automatically annotates all monoisotopic peaks. Bruker's SNAP algorithm allows the information of a whole isotope pattern to be used for a high precision annotation. An internal or external calibration is performed and adducts or background signals are removed before the peak list is submitted to a sequence library search. The calibration mode, the signal-remove information as well as the library search parameters (which library to search, the applied proteolytic enzyme, mass accuracy etc.) are included in a package that is specified in the Excel based sample spreadsheet described above. Typically, the search is done in real time during the batch acquisition and, should this be necessary, further processing is performed manually on a Windows NT system. The data that result from the automatic library searches are stored as HTML files and can later be visualized. Manual post-processing of "difficult samples In our laboratory, the MASCOT search software (Matrix Science Ltd.), which is a further developed version of the MOWSE algorithm, has proven to be excellent in terms of speed, search quality (probability based scoring parameters) and also the ability to find reliable, correct results from uninterpreted MS/MS data. Searches on the Intra- as well as the Internet are supported. We have implemented MASCOT searches in our software MSoBioTools that allows peptide map analysis (Fig. 7) as well as an extended analysis of MS/MS data and search results. Fig. 7: The Internet query leads to a peptide map search result in HTML format that contains a probability score, a sequence coverage, literature information about the top search candidates etc. Bruker Daltonik GmbH

4 2nd Round Analysis: PSD MALDI-TOF mass spectrometry is not only a powerful tool for accurate molecular weight determination of digest peptides. In addition, MALDI is extremely useful for MS/MS analysis (PSD or Post Source Decay), which is another means to protein identification if the map is not specific (Fig. 8). Depending on the results, PSD data of selected digest peaks could be used in such analytical situations: 1. Confirm successful identifications. 2. Identify or characterize peaks in spectra, which remained uninterpreted. 3. Unsafe assignments prompt the need for further confirmation. This is typical in the case of (a) very low protein amounts, (b) protein mixtures, or (c) low number of cleavage sites. Peptides masses already attributed to a protein could be removed from the list and the remaining ones being used either for a second search or they could be PSD analysed for confirmation or identification. 4. In case of a failed identification, several peaks can be selected subsequently and PSD spectra acquired automatically for a "second path search. Fig. 8: PSD data can be submitted to a library search without any preinterpretation (top, left). MASCOT search results are furnished with a probability score for a convenient quality judgement (top, right). MS BioTools matches the PSD data to the search candidates, thereby allowing a complete interpretation of the results (bottom). Bruker Daltonik GmbH

5 Conclusion Proteomics is a synonym for the demand of a high quality, high throughput analytical technique. In combination with automatic sample preparation (MAP II), a powerful fuzzy logic based acquisition module (AutoXecute) and real-time data analysis (SNAP, MASCOT), MALDI-TOF MS is the method of choice for proteomics tasks of today and even more of the future. Together with automatic acquisition of PSD spectra currently being developed, 2nd round PSD experiments in automatic batch runs seem to be a very attractive strategy for high throughput protein identification. At present, around 80 % of the proteins in a 2D gel can be identified using peptide maps only (depending on many factors, e.g., gel quality, database comprehensiveness). With PSD, another 5-10 % of the proteins can be identified. Remaining identifications either require searching at a later date (waiting for larger sequence libraries) or using Edman or ESI-MS/MS microsequencing (Fig. 9). With an increase in size and quality of the protein sequence libraries more and more proteins will be identifiable without additional sequencing. Furthermore, sophisticated software modules will take care about the post-translational modifications, which currently disturb routine analysis. Parallelisation of digestion, sample preparation and sample analysis will make MALDI-TOF a tool unbeatable in speed for many hundreds of protein measurements per day. High Throughput Confirm ORF Confirm Modifications End 2D Gel MS Fingerprint Library Search Identification + - known? Microsequencing: direct analysis: PSD-MALDI-TOF after protein isolation: ISD Gene targeting Fig. 9: Strategy for protein identification in proteomics projects. Acknowledgments We thank Ole Jensen and Matthias Mann (Odense University) for their support establishing the fuzzy logicbased acquisition software. References 1. D. Suckau, K.-O. Kräuter, U. Rapp, M. Mann, O. Jensen, Proceedings of the 45th ASMS Conference, Palm Springs, CA, June 1-5, 1997, O.N. Jensen, P. Mortensen, O. Vorm, M. Mann, Anal. Chem. 69 (1997), For more information, please visit our Worldwide Web sites on the internet, call your local Bruker sales representative, or contact us at one of our application centers. Europe Bruker Daltonik GmbH Fahrenheitstr. 4 D Bremen Tel +49 (421) Fax +49 (421) sales@bdal.de USA Bruker Daltonics, Inc. Manning Park Billerica, MA Tel +1 (978) Fax +1 (978) ms-sales@daltonics.bruker.com Bruker Saxonia Analytik GmbH Permoserstr. 15 D Leipzig Tel +49 (341) Fax +49 (341) sales@bsax.de Bruker Daltonik GmbH