Issue 4 Summer 2002 - Applied Biosystems

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18 new product review Pro ICAT Software Application For Automatic Interpretation of Proteomic Data P ro ICAT is a software application that works in conjunction with BioAnalyst software. Its function is to process LC/MS and LC/MS/MS Information Dependent Acquisition (IDA) data that has been acquired from ICAT reagent-labelled proteomics samples using an API QSTAR ® Pulsar LC/MS/MS System. The software identifies and quantifies proteins present in complex samples following labelling with ICAT reagents. Identification is performed with the new Interrogator search algorithm, which can perform extremely fast database searches. Quantitation is performed using the unique three-dimensional LC/MS Reconstruct algorithm. The system stores all results in a back-end relational database for easy, flexible access, with the capability to create a second, expression-dependent MS/MS analysis from the quantitation results. The software provides fast, accurate interpretation of data and lets the user directly compare results to raw data in BioAnalyst software. Key Features ➜ Automated processing of proteomics data for protein identification and quantitation ➜ Designed to enhance the ICAT reagent technique from Applied Biosystems, for quantitative protein expression analysis ➜ Performs extremely fast database searches for protein identification using the exclusive Interrogator search algorithm, which accommodates as many as five variable modifications and uses a powerful ‘zone modification’ feature ➜ Unique LC/MS Reconstruct quantitation algorithm performs accurate three-dimensional determination of experimental:control ratios (D8:D0) ➜ Uses a back-end relational database for storing results ➜ Lets the user create expression dependent LC/MS/MS methods from quantitation results ➜ One click allows visualisation of quantitative evidence, protein sequences, and identification evidence with familiar BioAnalyst software tools Overview Traditional mass spectrometry approaches for protein identification involve separating protein mixtures using 1-D or 2-D gels, isolating the protein spots of interest, enzymatically digesting the protein into peptides, and analysing the peptides by mass spectrometry. The recent development of the isotopecoded affinity tag technique 1 has allowed the investigation of large numbers of proteins, using a multi-dimensional liquid chromatography approach (MDLC) in a fraction of the time typically necessary for traditional 2-D gel techniques. The ICAT reagent technique is based upon the use of a specific reagent that selectively modifies cysteine residues. Once modified, the proteins from two samples are combined and digested. By taking advantage of a biotin affinity tag on the reagent, the peptides containing the modified cysteine residues are selectively purified 2 . Once purified, ICAT reagent-labelled cysteine-containing peptides are separated by on-line capillary LC and analysed directly by mass spectrometry. Pro ICAT software is designed to identify and quantify ICAT reagent labelled peptides from LC/MS/MS IDA data acquired using an API QSTAR Pulsar Hybrid LC/MS/MS System. The instrument automatically performs MS and MS/MS on the eluting peptides using IDA3. The Pro ICAT software application processes the IDA data to identify proteins from the MS/MS spectra and quantify ICAT reagent-labelled expression pairs from the MS data. If desired, the IDA method can be generated from the quantitation results from an LC/MS run. Thus, expression dependent MS/MS analysis can be performed on only those peptides with ratios that meet user-defined limits. Conclusions A new software application is now available for processing proteomics data acquired from an API QSTAR Pulsar LC/MS/MS System, from samples prepared using the ICAT reagent technique. Pro ICAT software uses the Interrogator search algorithm for identifying proteins from un-interpreted MS/MS data and the LC/MS Reconstruct algorithm for quantitating expression pairs from the MS data. Users can define as many as five modifications in the protein identification search. Additionally, they can search for arbitrary modifications up to a user-specified mass difference at run-time (zone modification feature). The LC/MS Reconstruct algorithm quantifies ICAT reagent expression pairs from MS data by clustering the data based upon ICAT reagent fragments and optimally collapsing adjacent spectra for a maximum signal-to-noise ratio. Users may also run expression dependent LC/MS/MS IDA experiments based upon the quantitation results. new product review When the goal is to quickly process proteomic data from ICAT reagent labelled samples from the QSTAR system data, Pro ICAT software is the answer. Pro ICAT software identifies and quantifies proteins with high confidence and offers the additional flexibility of performing expression dependent analysis from interesting ratios found in the data. In summary, Pro ICAT software delivers the most comprehensive information in the shortest time and with the highest confidence to find the proteins that matter. References 1. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. 1999. Quantitative analysis of complex protein mixtures using isotopecoded affinity tags. Nature Biotech., 17:994-999. 2. For more information about the ICAT reagent technique, see the ICAT reagent product bulletin “ICAT Reagents for Quantitative Protein Expression Analysis Studies” (literature code: 125PB01-01). 3. For more information about IDA, see the LC/MS product bulletin “Information Dependent Acquisition – The Next Generation of Data Dependent Experiments for LC/MS/MS Analysis” (literature code: 114PB07-01). For more information on: Pro ICAT Software enter: No. 414 ICAT Reagents enter: No. 415 19
20 new product review Curtain Gas Interface The New Q TRAP LC/MS/MS System for Protein Analysis A Skimmer Orifice Q0 High- pressure Cell ST Q1 t the recent ASMS conference held in Orlando, Florida, Applied Biosystems/MDS SCIEX launched a new linear hybrid ion trap mass spectrometer, the Q TRAP LC/MS/MS System. This new Q TRAP LC/MS/MS technology combines the specificity and robustness of triple quadrupole systems with the full scan MS/MS sensitivity of ion trap systems into a single instrument. By combining these capabilities, improvements in both sensitivity and information content are realised. Additionally, the instrument has several unique scan modes, such as the ability to enhance the detection of multiply charged peptide ions over singly charged ions, mainly due to chemical noise introduced with a nanospray or LC interface. These unique features, in combination with acquisition tools such as Information Dependant Acquisition (IDA), make the Q TRAP LC/MS/MS System ideal for rapid and efficient identification of protein digests. Enhanced Sensitivity The Q TRAP LC/MS/MS System provides superior sensitivity in MS and MS/MS modes over that of traditional triple quadrupole and ion trap mass spectrometers while maintaining triple quadrupole-like fragmentation. With signal to noise improvements greater than 100X, identification of protein digests at femtomole levels can routinely be achieved with maximum sequence coverage. Enhanced Resolution The Q TRAP LC/MS/MS System provides superior resolution across the entire mass range, making charge state identification much more reliable up to charge state 5. This capability improves the ion selection process for complex mixtures and ensures the appropriate determination of collision energy in Q2 LINAC Collision Cell IQ2 IQ3 Q3 EXB C2B DF DET IDA mode. The Q TRAP LC/MS/MS System provides constant peak widths (0.12amu FWHH) across the mass range, thus resulting in resolution in excess of 4000 and mass accuracies of 50ppm for tryptic peptides selected by IDA for MS/MS. Enhancing Multiply Charged Ion Selection When maximum sample information is desired, it is extremely important to optimise the data acquisition for ions that will generate the most useful information. For protein digests at low levels, detection of multiply charged ions is normally hindered by the presence of background ions that are predominantly singly charged ions and usually present at high concentration. The Q TRAP LC/MS/MS System offers a unique operating mode where multiply charged ions are selectively detected over singly charged ions. This mode of operation ensures easy and efficient detection of peptide ions in both nanospray and capillary LC/MS mode, thus maximising the collection of useful information in a minimum amount of time. Information Dependent Acquisition (IDA) IDA enables the user to combine the unique modes of operation of the Q TRAP LC/MS/MS System to acquire MS and MS/MS data in an automated fashion within a single injection, thus maximising both instrument usage and information gathering. This can be achieved by combining modes of operation that yield very specific MS information such as precursor ion scans or multiply charged scans for proteolytic digests, with the enhanced full scan MS/MS mode of the Q TRAP LC/MS/MS System. new product review IDA provides enough flexibility to the user to combine scan types that will yield the most specific information at the highest quality level. Automated identification with ProID ProID is an application that works in conjunction with BioAnalyst software, and is designed to identify proteins from LC/MS/MS data acquired using Applied Biosystems/MDS SCIEX systems and information dependent acquisition (IDA). Identification is performed with the Interrogator search algorithm capable of performing extremely fast database searches with up to five variable modifications plus a powerful ‘zone’ modification feature for detection of unanticipated modifications. All results are stored in a back-end relational database with the ability to directly compare results to raw data in BioAnalyst software. The Q TRAP LC/MS/MS System is a cost efficient and reliable instrument for the identification of proteins. Its productivity, selectivity and sensitivity make it the instrument of choice for any proteomic laboratory that is looking for versatility. For more information on: Q TRAP LC/MS/MS System enter: No. 416 21
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Issue 4 Summer 2002 - Applied Biosystems

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