16
Figure 5. Product ion mass spectra of a mixture of HOBP and PEG 400 (1:20 molar ratio): (a) in-source CID without FAIMS separation; (b) FISCID-MS of selected HOBP ion (CV = 0.6-0.7V). (Reprinted (adapted) with permission from reference [2]. Copyright 2012, American Chemical Society).
The potential of using FISCID-MS for peptide identification was tested using peptide standards. A peptide mixture containing bradykinin, tetrapeptide MRFA, leucine enkephalin (LeuEnk), bombesin, and leutenising hormone releasing hormone (LHRH) produced complex mass spectra with multiple charge states for each peptide. Isolation of [Bradykinin+2H]2+ from the other singly and multiply charged peptides by FAIMS selection prior to in-source CID resulted in the identification of 21 characteristic bradykinin fragments due to significantly simplified fragment data, compared to just 6 without FAIMS pre-selection (data not shown).
The quantitative performance of LC-FISCID-MS was evaluated by spiking gramicidin S, not present in human plasma, into a human plasma tryptic digest. The relative standard deviation (RSD) for the precursor ion [gramicidin S+2H]2+ (9 ng on-column, m/z 571, n=6) was 5.1%, and less than 15% for the fragment ions (m/z 311, 424, 685 and 798) generated by FISCID-MS. The gramicidin precursor and product ions gave a linear response in the range 0.45-9 µg/ml for gramicidin S.
The LC-FISCID-MS method was also applied to the analysis of a complex mixture of tryptic peptides derived from depleted human plasma proteins to test the ability for peptide identification in a real sample. LC-MS of the plasma sample shows the presence of many components (Figure 6.a) resulting from the co-elution of tryptic peptides. The ion, m/z 480.8, at retention time of 3.5 min co-elutes with other compounds (Figure 6.b), which are observed in the mass spectrum taken across the SIC m/z 480.8 peak at half height (Figure 6.c). FAIMS selection (CV 2.5-2.6 V) removes the interfering ions (e.g. m/z 564 and 707) from the mass spectrum (Figure6.d). Product ion peaks from LC-CID-MS (Figure 6.e) and LC-FISCID-MS (Figure 6.f) were used for peptide identification. LC-FISCID-MS provided a simpler product ion spectrum with fewer but more prominent peaks. Peptide identification was carried out via the MASCOT search engine [7], using the SwissProt protein database. No significant hits were yielded from the analysis without a FAIMS separation, however LC-FISCID-MS gave human serum albumin (HSA), present at <0.4% in the depleted serum, as the top hit (confidence score 34, 27 or above deemed significant). The enhancement in selectivity, reproducibility and linear response shows that LC-FISCID-MS has the potential to offer significant benefits over a single mass analyser.
References
1. L.J. Brown, D.E Toutoungi, N.A. Devenport, J.C. Reynolds, G. Kaur-Atwal, B. Boyle, C.S. Creaser, Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis. Anal. Chem. 82 (2010) 9827
2. L.J. Brown, R.W. Smith, D.E. Toutoungi, J.C. Reynolds, A.W.T. Bristow, A. Ray, A. Sage, I.D. Wilson, D.J. Weston, B. Boyle, C.S. Creaser, Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry. Anal. Chem. 84 (2012) 4095
3. R.W. Smith, D.E. Toutoungi, J.C. Reynolds, A.W.T. Bristow, A. Ray, A. Sage, I.D. Wilson, D.J. Weston, B. Boyle, C.S. Creaser, Enhanced performance in the determination of ibuprofen 1--O-acyl glucuronide in urine by combining high field asymmetric waveform ion mobility spectrometry with liquid chromatography-time-of-flight mass spectrometry. J. Chrom. A 1278 (2013) 76
4. R.W. Smith, J. C. Reynolds, S.-L Lee, C.S. Creaser, Direct analysis of potentially genotoxic impurities by thermal desorption-field asymmetric waveform ion mobility spectrometry-mass spectrometry. Anal. Methods 5 (2013) 3799 5. T. McGovern and D. Jacobson-Kram, Regulation of genotoxic and carcinogenic impurities in drug substances and products. Trends Anal. Chem., 2006, 25, 790 6. B.A. Olson, B.C. Castle, D.P. Myers, Advances in HPLC technology for the determination of drug impurities. Trends Anal. Chem., 2006, 25, 796 7. Mascot search engine,
http://www.matrixscience.com, date accessed: 27/10/2011
Figure 6. LC-MS and LC-FISCID-MS analysis of human plasma tryptic digest: (a) TIC, (b) selected ion chromatograms at 3.4-3.6 min, (c) LC-MS spectrum of peaks at 3.52 min without FAIMS separation, (d) LC-FAIMS-MS spectrum with FAIMS selection of the m/z 480 ion (CV 2.5-2.6 V), (e) LC-in-source CID-MS spectrum without FAIMS selection, and (f) LC-FISCID-MS spectrum with FAIMS selection of the m/z 480 ion and in-source CID (CV 2.5-2.6 V). (Reprinted (adapted) with permission from reference [2]. Copyright 2012, American Chemical Society).
Conclusion
The integration of a miniaturised chip-based FAIMS device with mass spectrometry has been shown to enhance analytical capabilities in a number of ways, including improving limits of quantitation by reducing chemical noise, increasing selectivity by removing sample complexity and by offering the ability to distinguish between isobaric ions. Rapid FAIMS separation can be combined with chromatographic techniques to enhance detection and in some cases can provide an alternative to LC. The additional separation stage can also provide pre-selection prior to fragmentation via in-source CID, enabling quasi-tandem experiments on a single-stage MS. These results suggest that FAIMS has the potential to become a versatile tool for enhancing mass spectrometry.
Acknowledgements
The authors thank Owlstone Ltd. and Loughborough University for financial support. We thank Owlstone Ltd and Agilent Technologies for the provision of instrumentation and technical support and AstraZeneca for provision of chemicals.
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