High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry

✍ Scribed by Marina, Anabel; García, Miguel Angel; Albar, Juan Pablo; Yagüe, Jesús; López de Castro, José Antonio; Vázquez, Jesús

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 203 KB
• Volume: 34
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/(sici)1096-9888(199901)34:1<17::aid-jms746>3.0.co;2-6

✦ Synopsis

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the o †-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy. This needed a careful optimization of the number of ions stored inside the trap (ion target parameter) and required spectrum averaging of many scans. A method is presented for the mathematical Ðtting of ZoomScan spectra to theoretical isotopic distributions, which allowed the mass determination of large peptides with more accuracy than that achieved by conventional deconvolution algorithms. A very simple on-line desalting conÐguration is also described which needed no external micro-high-performance liquid chromatographic pumps, and can be easily mounted using the built-in syringe delivery system of the LCQ. This set-up allowed extended analysis times of "in-gelÏ protein digests in subpicomole amounts. Finally, the multiple fragmentation capabilities of the ion trap were found to be extremely useful for the analysis of peptide modiÐcations such as phosphorylation and for sequencing individual peptides from highly complex MHC-bound peptide pools.