High accuracy mass measurement of peptides with internal calibration using a dual electrospray ionization sprayer system for protein identification

Abstract

A dual-ESI-sprayer system was constructed and applied to achieve high accuracy of peptide mass measurement for protein identification by means of peptide mapping. Sample was introduced in one sprayer, and reference in the other, thus making internal calibration possible greatly enhancing the mass accuracy. Several samples were utilized to evaluate the reliability of this dual-ESI-sprayer system. The range of mass errors was 0.16-5.37 ppm. The peptide masses of tryptic digests of myoglobin (horse) were measured by the HPLC/dual-ESI-MS system, with mass deviations ranging from 0.01-7.67 ppm, and about 75% mass deviations below 5 ppm with 40% below 1[?]ppm. These peptide masses were utilized to perform database searching for protein identification, and compared to results obtained by external calibration. This comparison showed that the internal calibration provides a more reliable method of protein identification, with a much smaller number of required peptides for matching, and with less CPU time consumed for database searching.

Pulsed Nano-Electrospray Ionization: Characterization of Temporal Response and Implementation with a Flared Inlet Capillary

“…As noted above the ion flight time is approximately 250 μsec and the time it takes to break the Taylor cone is less than 50 μsec. [17,23,24] These results indicate that the majority of the decay time is in stopping the ion flux. The observed decay time is much longer than some reports found in the literature, which suggest decay times of less than 100 μsec.…”

“…[24,25] However, these reports measured the current directly on an electrode that immediately follows the nano-ESI or ESI needle. [17,23,24] Additionally, the solutions that were used in determining decay times were pure solvents, high concentration salts, or contained glycol. The ion currents that are reported in the literature are in the 10 to 100 nA region which may be the result of solvent clusters and is much greater than the ion currents expected from desolvated analyte ions.…”

“…Despite the large difference in decay times, reports that monitor the ion current in a pulsed electrospray system using a lens show that the trailing edge of the ion signal has a long decay time relative to the rise time. [17,23–25] In one report, time resolved photos show that ion signal is still present following the termination of the Taylor cone suggesting that charge in the spray solution takes some additional amount of time to dissipate and finally stop ion formation/desolvation. [25] In low flow rate systems, such as the ones in our experiments, the shape of the ion plume can become a mist which is possibly the environment that is created after the Taylor cone has dissipated but while there is still residual charge in the spray.…”

“…In addition to increasing throughput, multiple ESI sources have been used in parallel arrangements to improve mass measurement accuracy, [4] reduce adverse affects from multiple solutions interacting prior to analysis, [4,17] and allow ion/ion reactions to be studied. [18–21] Work has been presented on the use of two nano-ESI sprayers operating continuously but at opposite polarities for ion/ion reactions.…”

A modified nanospray tip coupled to a mass spectrometer for analyzing enzymatic peptides

“…Standard proteins were digested with trypsin in‐solution and in‐gel as described 18,19. A sample containing 500 ng of total cell lysate of human U937 monocyte was subjected to 2D gel separation according to previous work in our laboratory 20.…”