Quadrupole protein identification

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

Figeys, D., Lock, C., Taylor, L., and Aebersold, R. (1998). Microfabricated device coupled with an electrospray ionization quadrupole time-of-flight mass spectrometer protein identifications based on enhanced-resolution mass spectrometry and tandem mass spectrometry data. Rapid Commun. Mass Spectrom. 12, 1435 — 1444. 

New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification (Andersen and Mann 2000). 

Blackburn, R. K. Moseley, M. A. Ill 1999. Quadrupole time-of-flight mass spectrometry a powerful new tool for protein identification and characterization. Am. Pharm. Rev., 2, 49-59. 

Andersen, J., Molina, H., Moertz, E. et al. (1998) Quadrupole-TOF hybrid mass spectrometers bring improvements to protein identification and MS/MS analysis of intact proteins. The 46th Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, p. 978. 

Chait, B. T. (2000). Rapidly switchable matrix-assisted laser desorption/ ionization and electrospray quadrupole-time-of-flight mass spectrometry for protein identification. J. Am. Soc. Mass Spectrom. 11, 493-504. 

Various forms of tandem mass spectroscopy (MS/MS) have also been used in the analysis of biomolecules. Such instruments consist of an ionisation source (ESI or MALDI or other) attached to a first mass analyser followed by a gas-phase collision cell. This collison cell further fragments the selected ions and feeds these ions to a second mass detector. The final mass spectrum represents a ladder of fragment ions. In the case of peptides the collision cell usually cleaves the peptides at the amide bond. The ladder of resulting peptides reveals the sequence directly [496]. Thus, tandem MS instruments, such as the triple quadrupole and ion-trap instruments have been routinely applied in LC-MS/MS or ESI-MS/MS for peptide sequencing and protein identification via database searching. New configurations, which have been moving into this area include the hybrid Q-TOF [498], the MALDI-TOF-TOF [499] and the Fourier transform ion cyclotron resonance instruments [500]. 

Wang, N. Li, L. Exploring the precursor ion exclusion feature of liquid chromatography -electrospray ionization quadrupole time-of-flight mass spectrometry for improving protein identification in shotgun proteome analysis. Anal. Chem. 2008,80,4696-4710. 

DCIM-MS has been coupled to liquid chromatography (LC) for analyzing complex peptide samples [36], Peptides eluting from the LC column were analyzed on an IMS-Q-TOF mass spectrometer. The ions generated from the source were accumulated in an ion trap and injected periodically into the drift tube. After mass analysis by the quadrupole, ions were subjected to collision-induced dissociation (CID) within an octopole collision cell and the product ions were analyzed by a TOF analyzer. Using this instrumental configuration, the urinary proteome [37], the Drosophila melano-gaster head proteome [38], and the human plasma proteome [39] have been analyzed. While many additional measurements, compared to standard mass spectrometry-based proteomics experiments, were obtained (for example, collision cross-sections), these were not used to improve upon protein identification results. 

Figeys, D. Aebersold, R. High sensitivity identification of proteins by electrospray ionization tandem mass spectrometry inital comparison between an ion trap mass spectrometer and a triple quadrupole mass spectrometer. Electrophoresis 1997,18, 360-368. 

Williamson, B. L., Marchese, J., and Morrice, N. A. (2006). Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer. Mol. Cell. Proteomics 5 337-346. 

The ESI-LC/MS-based approaches that feature ion trap (Gatlin et al, 1998 Washburn et ah, 2001) and quadrupole time-of-flight (QTOF) (Blackburn and Moseley, 1999) mass spectrometers are routinely used for the identification and characterization of proteins. Nanoelectrospray LC/MS formats (Figure 6.3) are used to provide lower limits of detection and fully automated sample preconcentration and desalting. On-line LC/MS approaches for protein expression profiling are also used with ESI-TOF (Banks and Gulcicek, 1997 Chong et al., 2001) and ESI-Fourier transform (FT) (Kelleher... 

Swiderek, K. M. Davis, M. T. Lee,T. D. 1998. The identification of peptide modifications derived from gel-separated proteins using electrospray triple quadrupole and ion trap analyses. Electrophoresis, 19,989-997. 

MALDI quadrupole ion trap mass spectrometry has also been used to localize and identify the post-translational modifications on the Sindai virus [18]. The polymerase associated protein (P protein) from this virus is reported in the literature to be highly phosphorylated. In vitro studies have detected phosphorylation in different regions of the protein, while a single phosphorylation site was found in the in vivo studies. Mass spectral data, along with computer-aided analysis, enabled the identification and localization of two phosphorylation sites. 

For the identification of low-spin iron(Il), a low value of the quadrupole splitting is generally not enough. As an example, the Mossbauer spectrum of the reduced low-spin ferrous form of cytochrome C552 is shown in Figure 10. The reduced heme iron has 5 = 0.46 nun s and AEq = -Fl.30mms . These values are characteristic for the low-spin ferrous forms of several electron transfer heme proteins like cytochromes c and... 

The methods for each study are divided into the initial protein separation step, a second separation step if applicable, the type of mass analysis, and the software used for peptide identification. ID = one dimensional polyacrylamide gel electrophoresis, 2D = two dimensional polyacrylamide gel electrophoresis, MS = mass spectrometry (peptide mass fingerprinting), MS/MS = tandem mass spectrometry, MALDI-TOF = matrix assisted laser desorption/ionization-time of flight, MS FIT = software from Protein Prospector, http //prospector.ucsf edu/, ESI = electrospray ionization, Q-TOF = quadrupole-time of flight, PPSS2 =Protana s Proteomic Software Suite (ProtanaEngineering, Odense, Denmark), Mascot = Matiix Science, http //www.matrixscience.com/, TOF-TOF = MALDI plus TOF tandem mass spectrometry, RP-HPLC = reverse phase high performance liquid chromatography, Q-IT = quadrupole ion trap, LIT = linear ion trap. Bioworks = Thermo Electron Corporation. 

Prefractionation of proteins in an Arabidopsis thalicma leaf extract by means of fast-performance LC prior to SCXxRPLC-MS on a monolithic RPLC column allowed the identification of 1032 unique proteins in 4 mg of a protein plant leaf tissue extract [59]. Fractionation of human plasma proteins by on-line SCX and RPLC was performed prior to tryptic digestion [60]. The resulting 30 fractions were digested and analysed by LC-MS on a quadrupole-linear-ion-trap (Q-LIT) instmment. Using this approach, 1292 proteins were identified, some of which are know to be present at <10 ng/ml. 

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