Trickle thinks ABI may be the winner at ASMS, but you wouldn't know it from the stock price. Maybe it was anticipation. Their new product launches are generating interest, the ISB guys are on the stump (Ruedi Aebersold mentioned I-CAT in his S.D. talk, lots of abstracts from them here, etc.) in part on ABI's behalf. Their analysis software is getting better (WAT is the leader here? WAT's frontend systems help in terms of one stop shopping, too). Their new TOF/TOF, though twice as expensive, is over five times faster. Here are a couple of abstracts in support of ABI's TOF/TOF effort:
>>Session: Proteomics - New Techniques Code: MOAam Time Slot/Poster Number: 11:55
Reliable Protein Identification from Stained or Unstained 2D gels using High Throughput MALDI TOF/TOF MS.
A.L. Burlingame1; Lan Huang1; Katherine E. Williams1; Carina Sihlbom1; Katalin F. Medzhiradszky1; Maria Pallavicini1; Jennifer M. Campbell2; Peter Juhasz2; Steven A. Martin2; Marvin L. Vestal2; Michael A. Baldwin1
1University of California, San Francisco, CA; 2Applied Biosystems, Framingham, MA
Novel Aspect: 2-D gel pixelation allows rapid, sensitive & comprehensive protein identification by MALDI-TOF/TOF
Introduction:
Mass spectrometry has become an essential tool for global deciphering of the human proteome. However, effective strategies for comprehensive identification of proteins from cultured cells, tissues or other biological samples remain a major challenge. In particular, highly sensitive methods are needed for proteins expressed in very low copy numbers. Using high sensitivity tandem MS, i.e. MALDI-TOF/TOF or nanoflow HPLC-QqoaTOF, we are exploring methods for routine sequencing and identification of peptides below the threshold for silver or fluorescent gel staining. Studies of primary tumor tissue place severe limits on total numbers of cells available. These preliminary studies were carried out using cells obtained from a promyelocytic leukemia cell culture.
Methods:
Standard protein identification by digestion and analysis of spots from a stained gel was compared with cutting an unstained gel into pixels and analyzing every pixel. To evaluate this, whole cell lysates from 5x106 NB4 acute promyelocytic cells were separated on 2-D gels. 100 pixels were cut from a representative 2 cm square of an unstained gel and visible spots were cut from the corresponding region of a 2nd gel stained with silver. All gel pieces were digested with trypsin and analyzed by MALDI-TOF to identify peptide masses for MS/MS. Subsequent high energy MALDI-TOF/TOF CID spectra were recorded on selected ions from the same sample deposits. Further aliquots of selected samples were analyzed by nano-HPLC/QSTAR. Tandem spectra were submitted to MS-Tag or sequenced manually.
Preliminary Data:
Due to low levels of expression and substoichiometric modification of many important proteins, complete pixelation of unstained gels may be advantageous. Here MS imaging of peptides in the pixelated region of an unstained gel identified 9 spots, only 5 of which were equivalent to silver-stained spots in a second gel. The remaining 4 all gave TOF/TOF data of sufficient quality for identification. Thus mass spectrometry identified proteins below the level of staining with silver (Sypro ruby gave staining detection similar to silver). This supports the concept of entire 1- or 2-D gel pixelation to provide improved dynamic range and comprehensive sequence information from limited amounts of material. However, because the cutting, digestion, extraction and cleaning of the entire pixelated gel is labor intensive and time consuming, the practical realization of such a strategy will require sample handling robotics. Also, rapid sample analysis based on the use of a high frequency laser plus further automation of the instrument operation, data acquisition and database searching will be essential to handle the large numbers of samples generated by gel pixelation. Meanwhile from these preliminary results the MALDI TOF/TOF mass spectrometer has established its ability to generate high energy CID spectra with low fmol sensitivity with sufficient speed to cope with such a strategy. Furthermore, most spectra were of sufficient quality to be suitable for de novo peptide sequencing, essential for dealing with unknown proteins or peptide sequences inconsistent with databases due to genomic sequence errors, differences in DNA splicing, chemical modifications or unpredicted enzyme cleavages. Although nano-HPLC/QSTAR analysis revealed additional peptides on analogous aliquots and provided high quality CID spectra, it was inherently slower. Therefore this will be restricted to the more conventional analysis of spots excised from stained gels. This work was supported by NIH NCRR 01614.
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Session: Proteomics - Fundamental Studies Code: MPG Time Slot/Poster Number: 158
High through put analysis of MALDI TOF/TOF and LCMS/MS data.
Richard J. Jacob1; Peter R. Baker1; Michael A. Baldwin1; Armin Graber2; A. L. Burlingame1
1University of California, San Francisco, CA; 2Applied Biosystems, Framingham, MA
Novel Aspect: A system is described for high throughput automated MALDI-TOF/TOF protein identification/analysis.
Introduction:
The emergence of proteomics as an important new field is based on the power of mass spectrometry to analyze and identify proteins more accurately and rapidly than previously, and with ever higher sensitivity. This, together with the completion of the human and several other genomes, is resulting in a huge increase in the production of raw data. Here we present a system currently being developed for the acquisition and management of large data sets. It combines automated spectral processing, batch-wise database searching and organization, and instrumentation control via feedback from the search results, then archiving and documentation of results. All sample information is maintained and managed by a laboratory information management system (LIMS). This work was supported by NIH NCRR 01614.
Methods:
MS and CID data are obtained from a MALDI-TOF/TOF analyzing several thousand samples per day or a nanoflow HPLC-QSTAR operated in information dependent acquisition (IDA) mode. The MALDI-TOF/TOF produces data very rapidly, the analysis of which is required for feedback control of subsequent analysis of the same sample spots. Control of the LC-MS/MS system via the vendor’s software produces a file of about 50 MB per chromatogram, containing several hundred MS and MS/MS spectra. Signal processing including chemical noise removal, isotope removal, centroiding, and assessment of quality is required for both systems before batch-wise submission to the ProteinProspector search engine. Data is archived and search results are stored in the LIMS.
Preliminary Data:
An evaluation of differential protein expression in cell lysates separated by 2-D gels may require analysis of many thousands of spots per gel, particularly to identify proteins below the level sensitive to staining. To achieve such analyses within a reasonable time, robotic sample preparation and automated MS operation, data analysis and interpretation are essential. Comprehensive and well organized sample management, archiving of data and results, such as can be achieved with a LIMS, are also critically important. A system is being created for high throughput MALDI TOF/TOF tandem mass spectrometry. For the TOF/TOF to operate at maximum capacity, spectra acquired in a first round of MS experiments are processed and analyzed and the results used to direct subsequent high energy CID experiments. A software agent running on a second computer (the manager) obtains and processes each spectrum from the instrument computer, using pre-defined functions for noise removal, smoothing, isotope removal and centroiding, to create a peak list. Groups of peak lists generated in a first pass of the sample plate spectra are batched and submitted to MS-Fit running on a separate Protein Prospector server. From mass mapping results, a new sample table is generated for a second pass of the plate with peptide masses chosen to verify the presence of matched proteins. High energy CID data is recorded, processed by the manager and searched in a similar fashion using MS-Tag. Peptide masses unrelated to the identified proteins are then used to generate the sample table for the next round of experiments. This continues until all samples on the plate have been analyzed. Alternatively for samples of the greatest interest, a nanobore-HPLC-MS/MS IDA analysis of a digest from a single spot yields more comprehensive analysis. The raw data is processed, including determination of the charge state of each isotopic peak cluster, and the picked peaks are submitted for batch-wise database searching.<<
Cheers, Tuck |
