Data-dependent acquisition

MS/MS Duty Cycle Typical MS/MS analysis is a serial process, relying on the selection of precursors (peptides) in MS mode, followed by high-energy fragmentation in MS/MS. This process is termed data dependent acquisition (DDA). The duty cycle for the completion of MS and MS/MS cycles (the time necessary for MS/MS spectrum acquisition) is of primary importance. When the separation performance is viewed from the mass spectrometry perspective, the peak capacity can be characterized by the number of MS/MS scans, yielding successful... 

Data-dependent acquisition ability has been developed and incorporated into most software packages [MetaboLynx, Xcalibur, and Analyst Information Dependent Acquisition (IDA)]. In data-dependent acquisition mode, a mass spectrometer decides on the fly whether to collect MS/MS or MSn data, remain in full scan MS mode, or conduct other survey scans based upon user-defined criteria. Product ion spectra of potential metabolites can be automatically acquired in a single LC/ MS run. However, false positives may be generated due to highly intense matrix ion signals that may inadvertently trigger MS/MS or MSn scan functions. 

To be able to use MS/MS spectra library searching for general unknown screening, it is necessary to use an automatic process, called data dependent acquisition or information-dependent acquisition, to select the parent ions of interest, totally unexpected by definition, and to dissociate them and monitor their fragments. 

When working with non-radiolabeled drugs the major challenge is to find metabolites in the biological matrices. Because the enzymes responsible for metabolism are quite well characterized metabolic changes can partially be predicted. For example hydroxylation of the parent drug is in many cases the principal metabolic pathway. From a mass spectrometric point of view it results in an increase of 16 units in the mass spectrum. In the full-scan mode an extracted ion current profile can be used to screen for potential metabolites. In a second step a product ion spectrum is recorded for structural interpretation. Ideally, one would like to obtain relative molecular mass information and the corresponding product ion spectrum in the same LC-MS run. This information can be obtained by data dependant acquisition (DDA), as illustrated in Fig. 1.39. 

In this case the survey scan was set as a full scan and the dependent scan as a product ion scan. The problem with data dependent acquisition is to determine the selection criteria. In most cases the system picks up the most abundant ion in the full scan spectrum. An inclusion list with masses of potential metabolites or exclusion list of known interferences significantly improves the procedure. In the example shown in Fig. 1.39, a procedure called dynamic background subtraction (DBS) was applied. This procedure considers chromatographic peak shapes and monitors not the most abundant signal in the spectrum but the largest increase of an ion in a spectrum. The advantage is that once a signal of a peak has... 

Either the information obtained during the data-dependent acquisition is sufficient or a fraction of interest can be re-analyzed by chip-based infusion at a flow rate ca. 200 nl min. Due to the miniaturization sample consumption is very low (typically 1-3 pi) and acquisition time is no longer critical. Therefore various MS experiments can be performed on various instruments, including MS and accurate mass measurements. An additional advantage is that the eluent can be removed and the infusion solvent can be optimized for positive or negative ion detection or for deuterium exchange measurements. 

A similar approach using accurate mass measurements and predictive fragmentation sofiware was also applied for the examination of the human microsomal metabolism of nefazodone using a linear ion trap-orbitrap hybrid mass spectrometer. Based on a single LC-MS run, using data-dependant acquisition, 15 metabolites of nefazodone could be identified in MS and MS/MS with a mass accuracy better than 3 ppm. 

Once peptides have been successfully eluted from multi-dimensional chromatography, they undergo CID within MS/MS instruments. Data-dependent acquisition systems associated with MS/MS allow individual peptides to be fragmented and the resultant spectra to be used to search the databases for possible identification (Wehr, 2003). This method of protein identification, via MuDPIT, is most efficient when working with genome-verified organisms (Yates, 1998). MuDPIT... 

Figure 11-16. A user-defined threshold for percent parent stability is pre-set in the custom software. Compounds dropping below this pre-defined threshold are automatically selected for detailed metabolite identification by LC-MS/MS incorporating data-dependent acquisition, parent, and precursor ion scans.

Schwudke D, Oegema J, Burton L, Entchev E, Hannich JT, Ejsing CS, Kurzchaha T, Shevchenko A. Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition. Anal. Chem. 2006 78 585-595. 

An important issue is the need to acquire both MS and MS-MS data for the unknowns. In general, this requires two injections with data-processing in between. The first run is done in full-spectrum LC-MS mode. Precmsor /w/z-values for relevant peaks in this chromatogram have to be determined in (manual) data processing. The /w/z-values found are then used in a time-scheduled product-ion MS-MS procedure using multiple precmsor-ions. Alternatively, data-dependent acquisition (DDA, Ch. 2.4.2) can be used, as demonstrated by Drexler et al. [106]. An alternative to DDA in a triple-quadrupole instrument is the RF product-ion analysis mode (RFD), proposed by Kienhuis and Geerdink [107]. 

Data-dependent acquisition (DDA) on an ion-trap instrument was applied in combination with in-source CID for the analysis of C-glycosyhlavone-O-glycosides in plant extracts [38], DDA controlled switching between a survey MS scan and MS-MS allowed automated LC-MS analysis of plant extracts. Differentiation between 6- and 8-C-glycosylation is made via the in-source CID generation of X-ions and subsequent MS-MS analysis. 

Yan, Z., Caldwell, G.W., and Maher, N., Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition, Anal. Chem., 80(16), 6410, 2008. 

Another strategy for the detection of glutathione adducts is to rely on cluster analysis approaches in which a labeled form and a nonlabeled form of glutathione are used at a defined ratio. Various labels (Table 6.1) have been proposed and Mutlib et al. [18] used a deuterium labeled in the glutamate moiety (mass difference 3 Da) and switched later to a GSH-labeled 13C2-15N in the glycine moiety (mass difference 3 Da) [19], If the data-dependent acquisition mode is positive in the survey scan, then the... 

---