Abstract
Electrospray ionisation (ESI) is renowned for its ability to ionise intact proteins for sensitive detection by mass spectrometry (MS). However, the use of a conventional direct current ESI voltage can result in the formation of relatively large initial droplet sizes, which can limit efficient ion desolvation and sensitivity. Here, pulsed nanoESI (nESI) MS using nanoscale emitters with inner diameters of ~250 nm is reported. In this approach, the nESI voltage is rapidly pulsed from 0 to ~1.5 kV with sub-nanosecond rise times, duty cycles from 10 to 90%, and repetition rates of 10 to 350 kHz. Using pulsed nESI, the performance of MS for the detection of intact proteins can be improved in terms of increased ion abundances and decreased noise. The absolute ion abundances and signal-to-noise levels of protonated ubiquitin, cytochrome C, myoglobin, and carbonic anhydrase II formed from standard denaturing solutions can be increased by up to 82% and 154% using an optimal repetition rate of ~200 kHz compared to conventional nESI-MS. Applying pulsed nESI-MS to a mixture of four proteins resulted in the signal for each protein increasing by up to 184% compared to the more conventional nESI-MS. For smaller ions (≤1032 m/z), the signal can also be increased by the use of high repetition rates (200–250 kHz), which is consistent with the enhanced performance depending more on general factors associated with the ESI process (e.g., smaller initial droplet sizes and reduced Coulombic repulsion in the spray plume) rather than analyte-specific effects (e.g., electrophoretic mobility). The enhanced sensitivity of pulsed nESI is anticipated to be beneficial for many different types of tandem mass spectrometry measurements.
Highlights
Electrospray ionisation (ESI) is well known for its ability to form intact protein ions for sensitive detection by mass spectrometry [1]
The use of high voltage and high frequency pulses in nESI-mass spectrometry (MS) can result in significant performance gains in terms of the signal intensity, background chemical noise (NC), and signal-to-background chemical noise (S/NC) in the detection of intact proteins compared to conventional direct current (DC) nESI-MS
The use of pulsed high voltage waveforms in nESI-MS can be used to significantly increase the abundances of protein ions formed from mixtures of proteins by up to 184% compared to DC nESI-MS
Summary
Electrospray ionisation (ESI) is well known for its ability to form intact protein ions for sensitive detection by mass spectrometry [1]. A key characteristic of ESI is the formation of a distribution of highly charged ions [2,3]. This multiple charging effect has many advantages. High charging extends the effective mass range of instruments with upper m/z limits, such that proteins can be detected on essentially any type of ESI-equipped mass spectrometer [4]. For charge-sensitive mass analysers, the instrument response increases linearly with the charge state of the ion and more highly charged ions can be detected with higher sensitivity and lower detection limits [5]. In electron capture dissociation (ECD) [6,7,8], electron transfer dissociation (ETD) [9,10], and some types of ultraviolet photodissociation (UVPD) [11,12], the extent of the ion dissociation and sequence coverage can increase significantly with both the charge state and the abundance of the precursor ion
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