Strategies for determination of insulin with tandem electrospray mass spectrometry: implications for other analyte proteins?

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Abstract
Using human insulin (MW 5808 Da) as a model compound, the possible strategies towards optimization of sensitivity and selectivity of measurement by electrospray ionization with a standard triple quadrupole mass spectrometer were investigated. For measurement in selected ion‐monitoring (SIM) mode, these strategies involved systematic variation of instrumental parameters and spray pH. In this investigation four different operating modes were used corresponding to positive/negative ionization modes with acidic/basic sprays and pH reversed (hereafter termed ‘wrong‐way‐round’ operation); the cone voltage was optimized for each mode of operation. When collision‐activated dissociation (CAD) is employed, two additional operation modes are possible: namely, low collision energies (10–35 eV, CAD‐l) for the generation of sequence‐specific fragments and high collision energies (>80 eV, CAD‐h) for the generation of nonspecific fragments. Overall, this results in twelve different modes of operation.Loop‐injection of aqueous insulin standards were run for each of the twelve operating modes and measurements made for five different charge states (n = 2–6) observable with our instrument that has an upper mass limit of m/z 4000. The signal/noise (S/N) ratio was optimized for each charge state, resulting in 60 measurements. The best S/N ratios (20 000) were achieved under positive SIM conditions with charge state 6 (m/z 969) and under ‘wrong‐way‐round’ negative SIM conditions with charge state 3 (m/z 1935). Lower S/N ratios were observed under positive CAD‐h conditions with charge state 5 (m/z 1163, S/N 15 000) and positive CAD‐l conditions with charge state 6 (m/z 969, S/N 10 000). All other operating modes gave maximum S/N ratios of 4000.For measurement of insulin standards, the results obtained show SIM to give the best S/N ratio. However, for samples in complex matrices, our general experience suggests CAD to be the preferable operating mode. Consequently, for the development of a quantitative method for proteins in general, it might be advocated that all of the twelve operating modes and all relevant charge states be investigated to find the optimum S/N ratio. Copyright © 2001 John Wiley & Sons, Ltd.
Funding Information
  • National Fund for Scientific Research (3G0001096)
  • Research Fund of the University of Ghent (BOF 01102096)

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