Microsoft powerpoint - 318_thursday_dindyal.ppt [read-only]

Implementing Information Dependent Acquisition to an Hybrid RF/DC Quadrupole- Linear Ion Trap Mass Spectrometer
Alina Dindyal, Jane Y. Zhao, Nic Bloomfield, J.C. Yves Le Blanc, Applied Biosystems|MDS Sciex, 71 Four Valley Drive, Concord (Ontario) L4K 4V8, Canada
OVERVIEW
Figure 2. IDA Detection of Buspirone Metabolites from Rat Liver Microsome
IDA for the Identification of Post-translational Modifications
Survey Scan (1)
Figure 1. IDA Decision
Survey Scan (2)
Using Precursor Ion Scans of 122 and 168.
Making Tree and Workflow
Information Dependent Acquisition Applications on QTRAPTM – a hybrid RF/DC quadrupole linear ion trap +EPI (402.25) Charge (+1): Exp 3, 6.891 min from Sample 1 (Buspirone Metabolites) of Buspirone.
identification of post-translational modification • Detection of phase I and II metabolites Criteria
of tryptic peptides [2, 3]. IDA on the QTRAPTM • Protein identification from a digest mixture • Identification of Post-Translational Modification selectivity of the precursor scan, the polarity Exclusion List
change capabilities and the high sensitivity of INTRODUCTION
the EPI scan. Precursor ion of 79 in negative Enhanced Resolution (ER)
ion mode was used as a survey scan to reveal The increasing importance of LC/MS in areas of drug discovery and proteomics has created a higher the presence of phosphorylated peptides, demand on the analytical capabilities of instrument in terms of sensitivity, throughput and automation. The followed by positive ER and EPI scans of the advent of tools like Information Dependent Analysis (IDA) enables user to improve their throughput by ion of detected (Figure 5). The ER scan has
Dependent Scan (s)
generating MS and MSMS data in a single analysis. However, in order to ensure that the information Dependent Scan (s)
the dual purpose of reporting the accurate generated will be useful, one has to either 1) design a high level of logic into the decision making process monoisotopic mass and assessing the charge MS3 Requested
Figure 3. IDA Detection Glucuronides of Buspirone Metabolites from Rat Liver
or 2) use instrument scans that will provide the desired information. The benefit of the former approach lies state of the peptide. With this information, the into the gathering of useful information based on the specificity of the analytical data generated. This can Microsome Using Neutral Loss Scans of 176
EPI scan was carried out in positive mode with be achieved efficiently if the mass spectrometer instrument can combine scan modes that provide specific Criteria
Hydroxy-Buspirone Glucuronide
MS information such as a triple quadrupole instrument with full scan MSMS sensitivity. The QTRAPTM EPI of 578
database search or de novo sequencing. MS3 of 578 → 402
hybrid quadrupole linear ion trap system brings together the power of instrument specificity and sensitivity with the automation level of IDA to maximize the collection of useful data to a plethora of applications – the Figure 5. α-Casein Digest Analysis with IDA
range of analyses performed spanned from discovery and toxicology to proteomics applications. This Second Dependent Scan (s)
CONCLUSIONS
poster presents the implementation and application possibilities of IDA on a Q TRAPTM system to Second Dependent Scan (s)
maximize data collection from single MS to MS3. In this poster we demonstrated how information dependent acquisition (IDA) performed on the prototype Q TRAPTM instrument yielded a wealth of information in applications spanning from Table 1. List of possible survey and dependent scans available on Q TRAPTM.
metabolite identification to proteomic analysis.
MATERIALS AND METHODS
The Enhanced Resolution scan, typical y used as a confirmation scan, could also be used as a Survey Scan. e32t0a3b
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Similarly, the EPI Scan could be used as a Survey Scan to automate the col ection of MS3 information.
the QTRAPTM instrument offers both the specificity of triple quadrupole scan types (neutral loss All the experiments were conducted on an hybrid RF/DC quadrupole linear ion trap mass spectrometer, IDA for the Identification of Proteins
and precursor ion) and the usual QTRAPTM full scan MSMS sensitivity on the same platform. This QTRAPTM (Applied Biosystems|MDS Sciex, Concord). Samples were introduced using either an Agilent flexible nature in combination with IDA allows the user to minimize the number of experiments capil ary LC system and a micro injector (Agilent, San Jose) OR a PE Series 200 micro pump and Identification of tryptic peptides from mixtures is challenging in many ways. This task can be made simpler required to obtain the right answer. The specificity of the survey scan also has the added benefit autosampler (Perkin Elmer Instrument, Norwalk) depending on the flow rate required for the application. by using the Enhanced Multiply Charged scan which enables the user to focus the collection of MSMS data of collecting only the useful information in the minimum amount of experiment, thus simplifying the Data collection and processing was done on Analyst® software with IDA.
on the relevant multiply charged precursor ions. Figure 4 shows the chromatogram and the corresponding
data review process. Specificity for proteomic applications can also be obtained with the Enhanced EMC, ER and EPI scans obtained. The ER scan reveals proper charge sate of the peptide and improved Multiply Charged scan of the QTRAPTM. The QTRAPTM functionalities allow users to devise IDA Description
the mass assignment, thus enabling the use of lower search tolerances and improving the search results.
experiments for their specific applications, with a high degree of accuracy and increased resolution. The result is an increased confidence in the results obtained, the ability to further refine In the Analyst acquisition software, IDA was implemented to offer flexibility while taking advantage of the Figure 4. Protein Identification Using IDA with One LC Injection
experiments for more thorough investigation together with a decrease in redundant data. scan specificity and sensitivity of MS systems such as QTRAPTM (Applied Biosystems|MDS SCIEX,
Concord, ON, Canada). The scan types available on QTRAPTM was listed in Table 1. IDA looped multiple
IDA for the Identification of Phase I and II Metabolites
REFERENCES
level experiments for the entire duration of the LC analysis (Figure 1). In order to take advantage of
E. H. Kerns, R.A.Rourick, K.J.Volk, M.S.Lee, Buspirone methabolite structure profile using a standard liquid specificity of scans such as precursor ion and neutral loss, while maximizing data collection, IDA has the The identification of metabolites in-vivo or in-vitro is frequently challenged by the presence of chemical noise chromatographic-mass spectrometric protocl, J. of Cromatography B, 698 (1997) 133-145
ability to combine the information from 2 survey scans in the decision making process. Enhanced or co-eluting endogenous species, especially under rapid chromatographic conditions. Though IDA can be H. Steen, B. Kuster, and M. Mann, Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the Resolution could be added as a confirmation scan to obtained MS information with higher resolution and used with inclusion list to focus on expected metabolite transformations, unexpected transformations wil determination of phosphopeptides by precursor ion scanning, JMS, 2001, 36: 782-790
improved mass accuracy. This information is particularly useful for charge state (z=1 to 5) determination remain undetected. Therefore, using precursor ion scan or neutral loss for phase I and II, respectively, can H. Steen, B. Kuster, M. Fernandez, A. Pandy, and M. Mann, Detection of tyrosine phosphorylated peptides by precursor and isotopic ratio measurements when used after scans (precursor or neutral loss) that may not reveal this ensure that unexpected metabolites can be more effectively detected and confirmed in a single injection. ion scanning quadrupole TOF mass spectrometry in positive mode, Anal.Chem. 2001, 73, 1440-1448
information. Since QTRAPTM offers the ability to collect MS3 data, a second level of dependency was Figure 2 shows the detection of Buspirone metabolites from rat liver microsome using precursor ion scans of
N. Pace, T. Gamble, J. C. Y. LeBlanc, Application of a Prototype Quadrupole-Linear Ion Trap Mass Spectrometer in the 122 and 168. All hydroxy-buspirone metabolites (9 by precursor of 122 and 2 by precursor of 168) previously Identification and Characterization of Glucuronides Metabolites, ASMS Poster #178, Thursday reported in the literature [1] were identified and confirmed by MSMS from a single injection in IDA mode. T. N. Gamble, N. Pace, J. C. Y. LeBlanc, Application of a Prototype Quadrupole-Linear Ion Trap Mass Spectrometer for enhanced resolution is carried out prior Among the criteria available for the IDA ion selection, SMART (specific mass and retention time) filters can the Quantitation of Metabolites in Biological Fluids, ASMS 2002 Poster #229, Wednesday.
Similarly, a neutral loss of 176 was used to identify 3 different glucuronides of buspirone metabolites. In be applied to the inclusion OR exclusion of ions of interest. This provides maximum flexibility to the user these experiments, MS3 was used to gain further fragmentation information on the aglicon species formed in when isobaric interferences are found or for rapid specific analysis when MSMS confirmation is required.
the collision cell. When compared to single MS as a survey scan, unless an inclusion list was used, we TRADEMARKS/LICENSING
could detect only a subset of the relevant metabolites of buspirone due to the weaker signals of the Q TRAP is trademarks of Applied Biosystems/MDS SCIEX. Applied Biosystems, Applied Biosystems (Design) and metabolite than that of other interferent ions eluting at the same retention times. Applera are trademarks of the Applera Corporation. MDS and MDS SCIEX are trademarks of MDS Inc.

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