Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.
Nepomuceno
Angelito I
AI
W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Muddiman
David C
DC
Bergen
H Robert
HR
Craighead
James R
JR
Burke
Michael J
MJ
Caskey
Patrick E
PE
Allan
Jonathan A
JA
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D002853:Chromatography, Liquid; D017357:Cyclotrons; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation*; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation*
D001059:Apoproteins; D009211:Myoglobin; D011506:Proteins; C010857:apomyoglobin