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Published in final edited form as: Int J Mass Spectrom. 2021 Mar 19;465:116578. doi: 10.1016/j.ijms.2021.116578

Application of frequency multiple FT-ICR-MS signal acquisition for improved proteome research

Sung-Gun Park 1, Jared P Mohr 1, Gordon A Anderson 2, James E Bruce 1
PMCID: PMC8059610  NIHMSID: NIHMS1692759  PMID: 33897275

Abstract

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with liquid chromatography (LC) is a powerful combination useful in many research areas due to the utility of high mass resolving power and mass measurement accuracy for studying highly complex samples. Ideally, every analyte in a complex sample can be subjected to accurate mass MS/MS analysis to aid in identification. FT-ICR MS can provide high mass resolving power and mass accuracy at the cost of long data acquisition periods, reducing the number of spectra that can be acquired per unit time. Frequency multiple signal acquisition has long been realized as an attractive method to obtain high mass resolving power and mass accuracy with shorter data acquisition periods. However, one of the limitations associated with frequency multiple signal acquisition is reduced signal intensity as compared to a traditional dipole detector. In this study, we demonstrated the use of a novel ICR cell to improve frequency multiple signal intensity and investigated the potential use of frequency multiple acquisition for proteome measurements. This novel ICR cell containing both dipole and frequency multiple detection electrodes was installed on a 7T FT-ICR MS coupled to an LC system. Tryptic digests of HeLa cell lysates were analyzed using dipole and frequency multiple detectors by holding either the mass resolving power or signal acquisition time constant. Compared to dipole detection, second frequency multiple detection yielded 36% or 45% more unique identified peptides from HeLa cell lysates at twice the scan rate or twice the mass resolving power, respectively. These results indicate that frequency multiple signal acquisition with either the same resolving power or the same signal acquisition duration as used with dipole signals can produce a significant increase in the number of peptides identified in complex proteome samples.

Graphical Abstract

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Introduction

Proteomics research is currently focused on the measurement of complex peptide mixtures from whole cell extracts, tissues or other samples. In such samples, there can be millions of unique peptides spanning a dynamic range in abundance beyond 106 creating unmet challenges for mass spectrometry scan rate coupled with chromatographic separations. On the other hand, mass measurement accuracy is crucial since tight tolerance on peptide and fragment masses can be highly discriminatory for proteome database searches. These two factors limit liquid chromatography with tandem mass spectrometry (LC/MS/MS) measurements of complex peptide mixtures and underpin the need for improved MS and MS/MS acquisition rates during chromatographic separation of proteome samples [1–4].

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) coupled with separation systems including LC [5–7], gas chromatography (GC) [8–10] and capillary electrophoresis (CE) [11, 12] is very powerful combination to analyze complex samples used in a variety of research areas, including proteomics. The achievable mass resolving power with FT-ICR MS is proportional to ICR signal transient length; longer transient lengths can yield higher resolving power. However, long data acquisition periods needed to acquire long transients limit the scan rate and the number of spectra that can be collected during a chromatographic separation. This limitation can be address in part by using stronger magnetic fields [13–17], multiple parallel mass analyzers [18, 19] and frequency multiple detectors [20–25].

As compared with dipole signal detection at the fundamental cyclotron frequency, frequency multiple detection can yield improved mass resolving power for equal data acquisition periods. Alternatively, frequency multiple signal acquisition can yield equal mass resolving power during a n-fold shorter signal acquisition period than that required for fundamental signal acquisition, where n is the order of the frequency multiple signals. Both could offer benefits for complex sample analysis and frequency multiple signals have been implemented in several research fields. For example, advantages of second [23, 26–28], third [29], fourth [20, 21, 29, 30] and sixth [25] frequency multiple detectors were demonstrated for intact analysis of proteins, crude oil and environmental contaminants. In all the cases, the ICR cells with nth order (n= 2, 3, 4 and 6) frequency multiple detectors showed n-fold higher mass resolving power or was observed for at the given acquisition period or n-fold faster data acquisition period at given mass resolving power as compared to traditional dipolar detectors.

However, the limitations associated with frequency multiple signal acquisition are undesired harmonic peaks and low harmonic signal amplitude as compared to the signals from a regular ICR cell with a traditional dipole detector [31]. The undesired harmonic peaks and low harmonic signal amplitude can make data interpretation more difficult and lead to identification errors. The undesired peaks can be minimized and/or simplified by modifying an ICR cell [32], direct comparison of parallel mass spectra obtained with the dipole and frequency multiple detectors for identical ions trapped in a ICR cell [30], or by applying offset voltages to detection and/or excitation electrodes[28, 33, 34]. The low harmonic signal amplitude can be improved theoretically by increasing excitation voltages that lead to larger cyclotron orbit radii for detection [21, 24, 30]. The larger cyclotron orbit radii can decrease the signal cancellation from the differential detection image current induced on adjacent plates [33]. However, increased excitation voltages can lead to increasing axial motion of ions and increasing the rate of dephasing of ion motion [31, 35]. Misharin and co-workers [29, 36] demonstrated higher harmonic signal sensitivity than that with a traditional dipole detector with ion cyclotron motion excited to half a cell radius using a coaxial multi-electrode cell (O-trap) that had internal coaxial electrodes around which the excited ions revolve and produce frequency multiple signals. In recent years, Nagornov and co-workers demonstrated a narrow aperture detection electrodes (NADEL) ICR cell at which detection electrodes were positioned radially inward of the ICR cell to detect unperturbed ion cyclotron frequencies [37, 38].

In this paper, we characterized the position of frequency multiple detection electrodes that were installed closer to the central axis of a ICR cell compared to a regular dipole detector for improved frequency multiple signal acquisition at low excitation voltages. The frequency multiple detector records sinusoidal transients with twice higher the frequency of a regular dipole detector during one period of ion rotation. After characterization, the benefit of frequency multiple signal acquisition in proteome analysis by LC/MS/MS was demonstrated by the comparison of results acquired with frequency multiple detection to those obtained with conventional fundamental frequency detection. To enable this experiment, a ThermoFisher Ultra ICR cell was modified to also include second frequency multiple detectors in addition to standard dipole detector electrodes. Tryptic digests of HeLa cell lysates were analyzed using fundamental and frequency multiple detection with otherwise identical conditions.

Experimental Section

LTQ FT-ICR MS.

A hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT-ICR MS; Thermo Scientific, Bremen, Germany) equipped with a 7 T actively shielded superconducting magnet (Jastec Japan Superconductor Technology, Tokyo, Japan) was used to acquire all experimental data. A cylindrical Ultra ICR cell in the FT-ICR MS was modified to support both dipole and frequency multiple detectors by replacement of entrance and exit lenses made with printed circuit board (PCB) on which copper wires were installed to serve as frequency multiple detectors. After installation of the modified ICR cell, the system was pumped down and baked out overnight. For initial tuning purposes with direct infusion a syringe pump flowed sample at 3.0 μL/min and an electrospray ionization (ESI) spray voltage of 4.8 kV was applied to a sample solution through a metal union to generate ions. Automatic gain control (AGC) was set to 3.0 × 104 for the ion trap and 5.0 × 105 for the ICR cell.

LC-MS.

To compare dipole and frequency multiple detectors for analysis of proteomic samples, tryptic digests of HeLa whole cell lysate sample were analyzed using a Water NanoAcquity UPLC (nanoACQUITY, Waters, Manchester, UK) coupled to LTQ FT-ICR MS equipped with a modified Ultra ICR cell that had both dipole and second frequency multiple detectors. The digested samples were separated using a 60cm × 75μm inner diameter fused silica analytical column packed with ReproSil-Pur C8 (5 μm diameter, 120 Å pore size particles). A mobile phase for LC consisted of water containing 0.1% (v/v) formic acid (solvent A) and acetonitrile containing 0.1% (v/v) formic acid (solvent B) and flow rate was 0.3μL/min. The linear gradient condition used for LC-MS analysis of samples was 90.0% solvent A (0.1% formic acid in water) and 10.0% solvent B (0.1% formic acid in acetonitrile) to 60% solvent A and 40% solvent B over 120 minutes at a flow rate 0.3μL/min. The mass spectrometer was operated in a data dependent acquisition mode (DDA) performing MS1 scan from 400 to 2000 m/z using FT-ICR followed by MS2 with LTQ on the 5 most abundant ions with 2+ and up inclusive detected in the MS1. The ions with 1+ and unassigned charge states in MS1 were excluded in MS2 scans. Parameters for MS2 scans included an AGC of 1.0 × 104, a maximum ion accumulation time of 100ms, an isolation window of 3.0 m/z, and a normalized collision energy of 35. A dynamic exclusion window of 30s was used to reduce redundant picking of the same parent ion. The obtained MS2 spectra from the LTQ were searched using Comet 2017.01 rev. 2. To compare the number of peptides identified with dipole and frequency multiple detectors, the same experiments as described above were repeated with different ICR data acquisition periods including 200ms and 100ms; a second frequency multiple detector theoretically can achieve twice the mass resolving power during the same data acquisition period and the same mass resolving power in half the data acquisition period as compared to a dipole detector. To obtain mass spectra with each detector, the mass spectra from a dipole detector with different data acquisition periods were obtained first, and then switched the detector to a frequency multiple detector. Before ICR signal acquisition with each detector, FT-ICR MS was calibrated with fundamental and second frequencies for dipole and frequency multiple detectors, respectively, using Met-Arg-Phe-Ala (MRFA) and Ultramark 1621 ions. To present mass spectra obtained with dipole and frequency multiple detectors, FT magnitude mode was used for both types of transients. In all LC-MS experiments, an ESI spray voltage of 2.5 kV was applied to sample solution through a platinum wire on PEEK micro cross, while other parameters were the same as those used in LTQ FT-ICR MS experiments as described above. The target samples for LC-MS experiments were measured three times for each acquisition time set-up. LC/MS data have been deposited into the PRIDE archive (https://www.ebi.ac.uk/pride/archive) with access number PXD022838.

ICR cell with dipole and frequency multiple detectors

In all experiments, dipolar detection and trapping electrodes in a ThermoFisher Ultra ICR cell were used with no modification. To include frequency multiple detectors for second frequency multiple signal acquisition, the original entrance and exit lens electrodes were replaced with entrance and exit lens plates made with PCB composed of FR4 substrate. Figures 1 and S1 show images of a modified Ultra ICR cell and entrance/exit plates. The diameter of entrance/exit plates was 47.2mm which contains entrance/exit lens electrodes with the diameter of 27.4mm and a 5.1mm diameter hole at the center of the electrodes through which ions from a LTQ were transferred into the modified Ultra ICR cell. The holes indicated with red rectangles and circles on Fig. 1b were used for installation of frequency multiple detection and excitation electrodes, respectively, that were constructed with 22 AWG copper wires. After installation of the PCB-based entrance/exit plates in place of the original entrance and exit lens electrodes, 22 AWG copper wires for frequency multiple detection and excitation electrodes were inserted into the holes and soldered to the PCB plates. The frequency multiple detection electrodes with the same polarity were connected by copper traces printed on the surface of the entrance/exit plates. To minimize shielding of the dipole detection electrodes, the frequency multiple detection electrodes were installed in front of original excitation electrodes used in the ThermoFisher Ultra ICR cell. The original excitation electrodes were replaced with copper wires to avoid distortion of the excitation field and minimize pre-amplifier saturation prior to detection. The original excitation electrodes were held at ground potential without their removal from the Ultra ICR cell. Each pair of frequency multiple detection electrodes consisted of two copper wires to obtain better signal-to-noise for second frequency multiple signals. Figure S2 shows the comparison of mass spectra obtained with second frequency multiple detectors that were made with 1 (Fig. S2a) and 2 (Fig. S2b) copper wires for each pair of frequency multiple electrode. The detection electrodes with one copper wire showed relatively lower signal intensities than those with 2 copper wires. The dipole and frequency multiple detectors were connected to individual vacuum feedthrough pins on a multiple-pin feedthrough flange using 24 AWG copper wires that were insulated with glass tubing as shown in Fig. 1a. The feedthrough pins on air-side were connected to the stock preamplifier for the ThermoFisher Ultra ICR cell. The other unused pins on the multi-pin feedthrough flange were held at ground potential. DC and RF voltages to trap and excite ions were applied to the ICR cell through another multi-pin feedthrough flange on the ion source side.[18, 19, 25, 30]

Figure 1.

Figure 1.

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Cylindrical ICR cell with PCB entrance and exit lens plates to install frequency multiple detection electrodes (a). Entrance and exit lens plates (b).

The entrance and exit lens plates were designed using the circuit board layout program EAGLE ver. 7.3.0 (CadSoft Computer, Pembroke Pines, FL) and manufactured by OSH Park (Advanced Circuits, Aurora, CO). The solder used for the ICR cell was 99.3/0.7 Sn-Cu lead-free solder alloy.[19, 39]

Trapping voltages were supplied by ThermoFisher power supplies to trapping electrodes as is normally done. ThermoFisher excitation waveforms as used with an Ultra ICR cell was used to excite the trapped ions. The mass range of m/z 200–2000 covering frequency range of approximately 500 kHz – 50 kHz was employed for excitation of ion. The excitation duration was 10ms. During acquisition of either fundamental or frequency multiple signals, the preamplifier was connected to dipole or frequency multiple detection electrodes and the other detection electrodes were held at ground potential.

Sample Preparation

Insulin, angiotensin, neurotensin and Ultramark 1621 (a mixture of fluorinated phosphazenes) were purchased from Sigma (St. Louis, MO, USA). HPLC grade methanol, acetic acid and dimethyl sulfoxide were obtained from Fisher Scientific (Pittsburgh, PA, USA). A 10 μM insulin standard solution was prepared by dissolving insulin in a 1:1 (v/v) water/methanol solvent mixture containing 0.1% (v/v) of acetic acid. An Ultramark 1621 stock solution was prepared by dissolving 10 μL of Ultramark 1621 in 10 mL of acetonitrile. A 10 μM solution of Ultramark 1621 was prepared by dissolving 100 μL of the stock solution of Ultramark 1621 in a solution of 1% acetic acid in 50:50 methanol:water. HeLa cells were grown to confluency in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% Fetalgro bovine growth serum (Rocky Mountain Biologicals) on a 15cm plate in an incubator maintaining an atmosphere of 37 °C and 5% CO2. Cells were harvested from the plate by incubation with 5mL of phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) with 20 mM EDTA. Following release, the cells were pelleted at 300g and then resuspended in a lysis buffer of 100 mM ammonium bicarbonate and 8M Urea. The cells were fully lysed by sonication using a GE-130 ultrasonic processor. The lysate was first reduced by adding TCEP to a final concentration of 5mM and incubating for 30 minutes, and then alkylated by adding iodoacetamide to a final concentration of 10mM and incubating for 45 minutes in the dark. The sample was then diluted 8-fold with 100 mM ammonium bicarbonate for a final Urea concentration of 1 M. Trypsin was added to the sample at a 1:200 (w/w) ratio to digest overnight at 37 °C. Finally, the resulting digest was desalted with a C18 sep-pak cartridge (Waters). The desalted peptides were resuspended to a final concentration of 1μg/μl in 2% acetonitrile containing 0.1% formic acid for mass spectrometry analysis.

Results and discussion

Characterization of the position of frequency multiple detection electrodes

To obtain high signal amplitude with a frequency multiple detector, the ions in an ICR cell need to be excited to larger cyclotron orbit radii than that with a regular dipole detector. However, higher excitation levels needed to produce a larger cyclotron radius can increase ion axial motion and the rate of dephasing of ion cyclotron motion [31, 35]. To minimize such adverse effects from increased excitation amplitude, frequency multiple detection electrodes were installed closer to the central axis of an ICR cell compared to regular dipole detection electrodes. Moving the frequency multiple detection electrodes closer to the central axis of the cell increases the frequency multiple signal intensity at relatively low excitation voltages typically used for a regular dipole detector. The dipole detection electrodes used in the ThermoFisher Ultra ICR cell were placed at 27.4mm from the central axis of the cell. The positions of frequency multiple detection electrodes for characterization were 21.3mm, 18.8mm, 16.2mm, 13.7mm and 11.2mm from the axis of the ICR cell which correspond to 80%, 70%, 60%, 50% and 40% radius of the dipole detection electrodes. Figure S1 show schematic of electrode assembly for frequency multiple detection electrodes installed at 16.2mm from the central axis of the ICR cell. Each position for the frequency multiple detectors was characterized using a 10 μM solution of MRFA and Ultramark 1621 at optimal trapping potentials. During ion injection, −9V was applied to entrance lens and front trapping electrodes, and +3V and 0V were applied to back trapping and exit lens electrodes, respectively. After ion injection and prior to excitation, the entrance lens and front trapping electrodes were switched to 0V and +3V, respectively, to trap ions. At 8ms after trapping ions, varied RF voltages were applied to excitation electrodes for 10ms to characterize ICR signals obtained with dipole and frequency multiple detectors. After excitation, the front and back trapping voltages were gradually decreased from +3V to +2V to obtain ICR signals. Figure 2 shows the fundamental and second frequency multiple signal intensities obtained with the second frequency multiple detector installed at different radii. In Fig. 2, red and blue bars indicate second (desired) and fundamental (undesired) frequencies of a MRFA ion, respectively, as measured with the second frequency multiple detector. The optimal excitation voltages used for second frequency multiple signal acquisitions with the second frequency multiple detector at 21.3mm, 18.8mm, 16.2mm, 13.7mm and 11.2mm away from the central axis of the cell were 59V, 54V, 49V, 44V and 35V peak to peak. The optimal RF voltages required to obtain second frequency multiple were decreased as the frequency multiple detector was installed closer to the center axis of the cell. However, the signal intensity for second frequency multiple increased as the frequency multiple detector was closer to the central axis of the cell until reaching 16.2mm. When the frequency multiple detector was installed at 13.7mm from the central axis of the cell, fundamental (undesired) signals showed 1.6 times higher intensity than second frequency multiple, and a 6-fold increase in a signal intensity with respect to the fundamental (undesired) frequency as compared to the frequency multiple detectors installed at 21.3mm, 18.8mm and 16.2mm radii. The frequency multiple detector installed at 11.2mm from the central axis of the cell led to a dramatical decrease in both signal intensities for fundamental (undesired) and second frequency multiples (desired). With the frequency multiple detector installed at 16.2mm radius, a 3-fold increase in a signal intensity for second frequency multiple (desired), but similar signal intensity for fundamental (undesired) frequency was obtained as compared to the frequency multiple detector installed at 21.3mm radius. The frequency multiple detection electrodes installed at 16.2mm from the central axis of the cell was similar to the detection electrode placement used in the NADEL cell design [34]. Figure S3 shows mass spectra obtained with dipole and frequency multiple detectors at optimal excitation voltages before and after installation of frequency multiple detection and excitation electrodes made with copper wires. Fig. S3a was obtained with a dipole detector at 38Vpp of excitation voltage using original dipole and excitation electrodes before modifying an Ultra ICR cell. Figs. S3b and c were obtained with the dipole and frequency multiple detectors, respectively, after installation of the frequency multiple detection and excitation electrodes. The frequency multiple detection electrodes obtained for Fig. S3c were installed at 16.2mm from the central axis of the cell. The optimal excitation voltages used to obtain the spectra were 46Vpp for the dipole detector (Fig. S3b) and 49Vpp for the frequency multiple detector (Fig. S3c) applying the excite waveform from excitation electrodes made with copper wires. The frequency multiple detector installed at 16.2mm from the central axis of the cell achieved similar signal intensity as compared to those with the dipole detectors shown in Figs. S3a and b.

Figure 2.

Figure 2.

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The intensities of fundamental (blue color) and second frequency multiple (red color) signals obtained with a second frequency multiple detector as a function of the frequency multiple detection electrodes being placed at various distances from the central axis of the ICR cell.

The relatively higher intensity of second frequency multiple peaks in mass spectra obtained with a second frequency multiple detector allowed an increase in mass spectra obtained with LTQ in DDA analysis. For example, the LC FT-ICR MS coupled with the second frequency multiple detector installed at 16.2mm radius acquired 60% more MS2 scans from the LTQ than the second frequency multiple detector installed at 21.3mm radius during the same data acquisition period (100ms). We believed that one of the reasons for the smaller number of MS2 in LC FT-ICR MS coupled with a second frequency multiple detector installed at 21.3mm radius was due to fundamental (undesired) peaks that showed relatively high intensity as compared to second frequency multiple peaks as shown in Figure 2 and Figure S2. During DDA analysis, the system will sometimes select fundamental (undesired) peaks for MS2 analysis due to their relatively high intensity. However, the targeted fundamental (undesired) peaks do not correspond a physical species, and consequently hit the maximum ion injection time which decreases the scan rate. Parallel detection of fundamental and harmonic signals is a possible solution to better utilize frequency multiple detection in future studies[25, 30]. In this technique, an ICR cell equipped with both a dipole detector and a frequency multiple detector is used to acquire simultaneous fundamental and frequency multiplied signals from a single ion population. The frequency multiplied signal is comprised of integer multiples of the fundamental frequency signal. The fundamental signals obtained with the dipole detector can therefore be used to decompose the complex mixture of signals from the frequency multiplied detector into its fundamental and individual frequency multiplied components.

Mass accuracy and mass resolving power

Mass accuracies for dipole and frequency multiple detectors were tested with the mixture solution of angiotensin, neurotensin, and insulin after calibration with MRFA and Ultramark 1621 that were injected with a syringe pump. The frequency multiple detection electrodes used for those tests were installed at optimal distance from the central axis of a cell (16.2mm radius), and the dipole detection electrodes were placed at 27.4mm radius. The excitation amplitude for dipole and multiple detection electrodes were 46Vpp and 49Vpp, respectively. As shown in Table 1, mass accuracies of less than 3 ppm were obtained from both detectors. The achieved mass resolving powers for dipole and frequency multiple detectors were evaluated with [M + 5H]5+ charge state insulin ions. The obtained mass resolving power from dipole and second frequency multiple detectors are shown in Figure 3. Figs. 3a and b are fundamental and second frequency multiple signals from the dipole and second frequency multiple detectors during 200ms and 100ms data acquisition periods, respectively. The second frequency multiple signals showed equal mass resolving power with 2-fold shorter data acquisition period as compared to the fundamental signals. Figure S4 shows another example of resolving powers for [M+4H]4+ ion (M= SHCIAEVEKDAIPENLPPLTADFAEDKDVCK) that was obtained from a tryptic digestion of BSA sample after separation with LC. The data acquisition times were 200ms and 100ms. As expected, the second frequency multiple detector (Fig. S4a) achieved 2 times higher mass resolving power as compared to the dipole detector with the same data acquisition time (Fig. S4b). Alternatively, the second frequency multiple detector (Fig. S4a) achieved the same mass resolving power as that with the dipole detector (Fig. S4c) during half data acquisition time.

Table 1.

Mass accuracy with dipole and frequency multiple detectors.

Ions m/z Dipole detector (ppm) Second frequency multiple detector (ppm)
[M+3H]3+
M = angiotensin		 432.8998 −2.7 −1.6
[M+3H]3+
M = Neurotensin		 558.3105 −2.0 1.7
[M+6H]6+
M = Insulin		 956.2752 2.1 −2.3
[M+5H]5+
M = Insulin		 1147.5295 −2.3 2.2
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Figure 3.

Figure 3.

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Mass spectra of +5 charged insulin ions with different data acquisition periods. Fundamental (a) and second frequency multiple signals (b) from dipole and second frequency multiple detectors, respectively. The data acquisition periods for the dipole and second frequency multiple detectors were 200ms and 100ms, respectively.

Online LC FT-ICR MS Application of a frequency multiple detector for analysis of proteins

To demonstrate the advantage of a frequency multiple detector in online LC FT-ICR MS used for proteome research, the peptides identified with the frequency multiple detector were compared to those with a dipole detector. To do that, 3μL of a HeLa cell lysates digest were injected into LC coupled with FT-ICR MS to acquire mass spectra using dipole and frequency multiple detectors during different data acquisition periods. Figure 4 shows a Venn diagram for peptides identified from a HeLa digest sample with the dipole and second frequency multiple detectors. Dipole detection enables identification of 6174 and 6548 unique peptides with 100ms and 200ms data acquisition periods, respectively. The number of peptides identified with second frequency multiple detection using a 100ms data acquisition period was 8915. This represented a 36% increase in the number of identified peptides as compared to dipole detection with equivalent mass resolving power and a 45% increase in the number of identified peptides as compared to the dipole detection with equivalent data acquisition time. Figure 5 shows the distribution of neutral masses identified with the dipole and second frequency multiple detectors. The second frequency multiple detector identified more peptides in the relatively low mass range as compared to the dipole detector at the same resolving power, but 2-fold shorter data acquisition time as shown in Fig. 5a. As compared to the dipole detector at the same data acquisition time, but 2-fold higher resolving power, the frequency multiple detector showed the increased number of identified peptides in relatively high mass range as shown in Fig. 5b.

Figure 4.

Figure 4.

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Venn diagram for peptides identified from a digested HeLa cell lysate sample with dipole and second frequency multiple detectors using 100ms and 200ms data acquisition periods. Blue and purple colors indicate the number of peptides identified with the dipole detector during 100ms and 200ms of data acquisition periods, respectively. Green color indicates the number of peptides identified with the second frequency multiple detector using 100ms data acquisition period.

Figure 5.

Figure 5.

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The distribution of natural masses of identified peptides obtained with dipole (blue color) and second frequency multiple (red color) detectors. a) Comparison of the number of identified peptides obtained with dipole and second frequency multiple detectors with equal resolving power. b) The comparison of identified peptides obtained with dipole and second frequency multiple detectors with equal data acquisition time.

CONCLUSIONS

In this study, we demonstrated the application of second frequency multiple signal acquisition for improved proteome studies. To improve the amplitude of second frequency multiple signals at relatively low RF voltages, the second frequency multiple detector was installed at 16.2mm from the central axis of a cell, that was 11.2mm closer to the central axis of the cell as compared to the dipole detector. At this condition, the second frequency multiple detector achieved the same intensity of second frequency multiple signals as compared to the dipole detector. The increased intensity of second frequency multiple peaks allowed an increased number of MS2 scans with an LTQ in DDA analysis because the improved signal intensity for second frequency multiple signals reduced the chance to select fundamental (undesired) peaks for fragmentation in the LTQ. In the analysis of a HeLa lysate with a second frequency multiple detector installed at 16.2mm radius, the second frequency multiple detector with 2-fold faster data acquisition time, but the same mass resolving power showed a 36% increase in the number of identified peptides. For the same data acquisition period, but 2-fold higher resolving power, the second frequency multiple detector achieved a 45% increase in the number of identified peptides from the HeLa sample. The results shown here illustrate that frequency multiple detection with novel ICR cells can result in a practical increase in the number of peptides identified with LC/MS/MS using standard DDA methods. Future efforts to improve on-the-fly-data processing that can effectively combine fundamental and frequency doubled signals will further increase signal-to-noise levels, improve selection efficiency and provide greater gains in terms of peptides identified per unit time. Additionally, our previous efforts have shown that frequency multiples up to 6 times the fundamental are possible with multi-detector electrode ICR cells. This advance, combined with higher magnetic field strengths that are currently possible will dramatically increase the effective scan rates for use with LC/MS/MS experiments using ICR-MS.

Supplementary Material

1

Figure S1. Schematic of electrode assembly for frequency multiple detection electrodes installed at 16.2mm from the central axis of a ICR cell.

Figure S2. Mass spectra obtained with frequency multiple detection electrodes that consisted of 1 (a) and 2 (b) copper wires. The peaks indicated with read color are second frequency multiple (desired) signals and the peaks indicated with blue color are fundamental (undesired) signals

Figure S3. Mass spectra obtained using different detectors; a) was obtained with a dipole detector at 38Vpp of excitation voltage before the installation of excitation and frequency multiple electrodes made with copper wires. b) and c) were obtained with dipole and second frequency multiple detectors at 46Vpp and 49Vpp of excitation voltages, respectively, after the installation of excitation and frequency multiple electrodes constructed with copper wires. The frequency multiple detection electrodes were installed at 16.2mm from the central axis of the cell.

Figure S4. Mass resolution of [M+4H]4+ (M= SHCIAEVEKDAIPENLPPLTADFAEDKDVCK) ion obtained with dipole and frequency multiple detectors for different data acquisition periods. (a) second frequency multiple signals using a 100ms data acquisition period. Fundamental signals from the dipole detector using 100ms (b) and 200ms (c) data acquisition periods.

Highlights.

  • Second frequency multiple detection increases scan acquisition rates or resolving power.

  • ICR cell modification was achieved to allow fundamental and second frequency multiple detection.

  • With LC/MS frequency multiple detection increased peptide identification by36% or 45% compared to conventional detection at equivalent resolving power or acquisition periods, respectively.

Acknowledgement

This work was supported by the National Institutes of Health through grant 5R01GM097112 and R35GM136255.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Supporting Information

Supporting Information materials available include: Supplemental Figures 1–4.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1. Schematic of electrode assembly for frequency multiple detection electrodes installed at 16.2mm from the central axis of a ICR cell.

Figure S2. Mass spectra obtained with frequency multiple detection electrodes that consisted of 1 (a) and 2 (b) copper wires. The peaks indicated with read color are second frequency multiple (desired) signals and the peaks indicated with blue color are fundamental (undesired) signals

Figure S3. Mass spectra obtained using different detectors; a) was obtained with a dipole detector at 38Vpp of excitation voltage before the installation of excitation and frequency multiple electrodes made with copper wires. b) and c) were obtained with dipole and second frequency multiple detectors at 46Vpp and 49Vpp of excitation voltages, respectively, after the installation of excitation and frequency multiple electrodes constructed with copper wires. The frequency multiple detection electrodes were installed at 16.2mm from the central axis of the cell.

Figure S4. Mass resolution of [M+4H]4+ (M= SHCIAEVEKDAIPENLPPLTADFAEDKDVCK) ion obtained with dipole and frequency multiple detectors for different data acquisition periods. (a) second frequency multiple signals using a 100ms data acquisition period. Fundamental signals from the dipole detector using 100ms (b) and 200ms (c) data acquisition periods.

NIHMS1692759-supplement-1.pptx (544.4KB, pptx)

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