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An Orthogonal Injection Ion Funnel Interface Providing Enhanced Performance for Selected Reaction Monitoring-Triple Quadruple Mass Spectrometry Tsung-Chi Chen, Thomas L. Fillmore, Spencer A. Prost, Ronald J. Moore, Yehia M. Ibrahim, and Richard D. Smith Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01482 • Publication Date (Web): 24 Jun 2015 Downloaded from http://pubs.acs.org on July 7, 2015

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Analytical Chemistry

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An Orthogonal Injection Ion Funnel Interface Providing Enhanced

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Performance for Selected Reaction Monitoring-Triple Quadrupole Mass

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Spectrometry

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Tsung-Chi Chen, Thomas L. Fillmore, Spencer A. Prost, Ronald J. Moore, Yehia M. Ibrahim and Richard D. Smith

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Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA

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Prepared for Analytical Chemistry

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Corresponding Authors: Yehia M. Ibrahim, E-mail: [email protected] Richard D. Smith, Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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The electrodynamic ion funnel facilitates efficient focusing and transfer of charged

3

particles in the higher pressure regions (e.g. ion source interfaces) of mass spectrometers,

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and thus provides increased sensitivity. An “off-axis” ion funnel design has been

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developed to reduce the source contamination and interferences from, e.g. ESI droplet

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residue and other poorly focused neutral or charged particles with very high mass-to

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charge ratios. In this study a dual ion funnel interface consisting of an orthogonal higher

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pressure electrodynamic ion funnel (HPIF) and an ion funnel trap combined with a triple

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quadrupole mass spectrometer was developed and characterized. An orthogonal ion

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injection inlet and a repeller plate electrode was used to direct ions to an ion funnel HPIF

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at 9-10 Torr pressure. Key factors for the HPIF performance characterized included the

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effects of RF amplitude, the DC gradient, and operating pressure. Compared to the triple

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quadrupole standard interface more than 4-fold improvement in the limit of detection for

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the direct quantitative MS analysis of low abundance peptides was observed. The

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sensitivity enhancement in liquid chromatography selected reaction monitoring (SRM)

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analyses of low abundance peptides spiked into a highly complex mixture was also

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compared with that obtained using a both commercial s-lens interface and a in-line dual

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ion funnel interface.

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Introduction

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The sensitivity of measurements using liquid chromatography with electrospray

3

ionization tandem mass spectrometry (LC/ESI/MS/MS) depends significantly upon the

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overall ion utilization efficiency1,2, including both the effectiveness of the ionization

5

processes and the efficiency of ion transmission from the source to the detector. The ESI

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efficiency for producing gas-phase ions is related to the solvent evaporation as well as

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repeated droplet fission3 that can take place at atmospheric pressure but also in the lower

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pressure regions, introducing both thermodynamic and kinetic constraints upon ion

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production.2,3

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Numerous approaches and interface designs for intermediate-pressure ion sampling

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and transmission devices4-7 have been developed to enhance ion transfer from

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sub-ambient pressure regions to the vacuum required for MS. The electrodynamic ion

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funnel has been shown to be broadly effective for the capture and focus of ions over a

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wide pressure range (30 Torr).5,8,9 Ion funnel designs generally utilize a stack of

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ring electrodes with gradually decreasing inner diameters. Ions travelling through the ion

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funnel are confined due to the radio frequency (RF) potentials of 180 degree phase

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shifted on adjacent electrodes, typically in conjunction with an auxiliary direct current

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(DC) field to drive ions through the ion funnel5,6 to focus ions though a conductance limit 3



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to the subsequent stages of the mass spectrometer. While ion funnels effectively transfer

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ions though sub-ambient pressure regions, they also have a modest focusing effect for

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larger particles and droplets, especially if entrained in a strong axial gas flow. The

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mixture of the ions and neutrals (e.g. from an electrosprayed solution) in a high collision

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rate environment can lead to additional gas phase chemistry that can impact

6

measurements (e.g. by proton transfer), as well as performance degradation due to the

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deposition on downstream ion optics. Problems become more pronounced as ions are

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more effectively transferred from ESI sources though multiple inlets in order to increase

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measurement sensitivity.10,11 Thus, the primary aims of “off-axis” ion introduction are to:

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1) reduce the interface and related ion optics contamination, and 2) decrease detector

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noise from excited and fast neutrals or very high m/z particles.12 The charged particles

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originating from the shrinking droplets are influenced by both gas dynamics and electric

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fields. Off-axis source concepts have been implemented, e.g. using ion funnels13,14,

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S-lens,15,16 conjoined ion guides,17,18 and bent RF-only quadrupole ion guides at

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intermediate pressure regions.12,19 The separation of the ions and neutrals within these

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devices can be facilitated by additional off-axis electrodes to obstruct neutral species and

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to produce fields that divert ions away from any directed gas flow.

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In the present study, we introduce an orthogonal ion funnel-ion funnel trap 4


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configuration on a triple quadrupole (QqQ) MS to improve robustness in conjunction

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with sensitivity. Comparing to conventional off-axis interfaces, incomplete desolvated

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droplets are unlikely to reach the exit orifice by the orthogonal ion funnel. While ions are

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sharply turned by 90 degree away from the direction of gas flow, the large acceptance

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area provided by the ion funnel maximizes the ion transmission efficiency. The reduced

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directed gas flow from the source and effective elimination of neutral particles can

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significantly improve both system robustness and detector signal-to-noise ratios. In this

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work the characterization of the orthogonal ion funnel in a triple quadrupole MS was

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evaluated and improved limits of detection (LODs) in both single stage MS and LC-SRM

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modes were achieved.

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Instrumentation

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The dual-stage ion funnel interface used in this work consists of a high pressure ion

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funnel (HPIF) and an Ion Funnel Trap (IFT)20,21 as shown schematically in Figure 1a.

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Ions generated in atmospheric pressure were introduced through either an inline (10 mm

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offset from the instrument centerline) or an orthogonal injection capillary. Following the

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HPIF is an IFT that can either trap or continuously transmit ions to the ion optics of the

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next stage. For the experiments presented in this manuscript the IFT was operated in the

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continuous (non-trapping) mode (i.e. functioning as a conventional ion funnel). As shown

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in Figure 1b, a mechanical pump connected to the HPIF chamber pumps out neutral

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species through an exhaust port and aligned with the expanding gas and charged particles

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from the injection capillary. Stainless steel capillaries with 1 mm i.d. and 10 cm long for

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both interfaces were chosen to increase the number of ions in conjunction with the HPIF

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chamber. The pressures of HPIF and IFT chambers were in the range of 8-10 Torr and ~1

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Torr, respectively, and a 2.5 mm i.d. conductance limit orifice followed the IFT allowing

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efficient optimal transmission to the triple quadrupole MS (TSQ Vantage, Thermo Fisher

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Scientific, San Jose, CA). The HPIF incorporated a repeller electrode that aided in

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directing ions towards the IFT that incorporated a stack of 46 Tin coated copper ring

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electrodes with 50.8 mm i.d. (i.e. the HPIF straight section in Figure 1) followed by a 6


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stack of 72 ring electrodes transitioning from an initial i.d. of 50.8 mm and decreasing

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gradually to a final electrode i.d. of 2.5 mm (i.e. the HPIF converging section). An RF

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waveform of equal amplitude but opposite phase was applied to adjacent ring electrodes

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in conjunction with a DC gradient to drive ion motion through the interface.

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Experimental

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Chemicals and Materials. For the initial tuning of the instrument, the triple

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quadrupole calibration stock solution (25 µM polytyrosine-1,3,6 in 1:1 water/methanol

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that contained 0.1% formic acid, Thermo Scientific Pierce, Rockford, IL) was used to

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optimize the DC gradient and RF amplitude applied to the HPIF and IFT. For quantitative

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analysis experiments, samples with different concentrations were prepared including: 1)

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The mixture of 9 peptides (Supplementary Materials Table S1) purchased from

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(Sigma-Aldrich, St. Louis, MO) was prepared at concentrations from 6 pmol/µL to 100

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nmol/µL in an electrospray solution of 1:1 water/methanol that contained 0.1% acetic

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acid by volume, and 2) synthetic peptides (Thermo Scientific, San Jose, CA) labeled with

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13

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carbonic anhydrase, bovine β-lactoglobulin, Escherichia coli β-galactosidase, and

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Prostate-specific antigen). Heavy peptides were estimated to be >95% pure, were spiked

C/15N on C-terminal lysine and arginine residues for the four target proteins (bovine

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into a tryptic digest of Shewanella oneidensis strain MR-1 to final concentrations ranging

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from 1 fmol/µL to 7.8 amol/µL. Detailed information on the target peptides is given in

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Supplementary Material Table S3.

4

LC-SRM Analysis. The prepared solution was analyzed with a nanoAcquity UPLC

5

system (Waters Corporation, Milford, MA) equipped with one analytical capillary

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column, 100 µm i.d.×100 mm with a 1.7 µm C18 (stationary phase, BEH130, Waters

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Corporation, Milford, MA), and an autosampler. Solvents used in the UPLC system

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consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in 90%

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acetonitrile (mobile phase B). Two µL of the solution for each concentration were loaded

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into the analytical column at a flow rate of 300 nL/min. The LC-SRM separation used a

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binary gradient of 10-15% B in 4.5 min, 15-25% B in 17 min, 25-38.5% B in 11 min,

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38.5-95% B in 1 min, and 95% B for 4 min.

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Nano-ESI Source. A chemically etched emitter22 of 20 µm i.d. was directly

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connected to a 75 µm i.d. capillary (Polymicro Technologies, Phoenix, AZ) for the MS

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analysis or to a LC analytical column through a zero volume stainless steel union (Valco

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Instrument Co. Inc., Houston, TX) for LC-SRM analysis. For the MS analysis, sample

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solutions were directly infused using a syringe pump (Thermo Scientific, San Jose, CA).

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Data Analysis. MS and MS2 data acquired using the TSQ Vantage were analyzed 8


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using an in-house developed data extraction program. The most abundant peaks (MS) or

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the three most abundant transitions (MS2) for each peptide (Tables 1 and 3) were used for

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quantification analyses. In the MS quantitation the LOD was defined as the lowest

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concentration point of target proteins at which the signal-to-noise ratio (S/N) of surrogate

5

peptides was >3 in a mass range of ±2 Da for the target peptides. In the LC-SRM

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quantitation the tandem MS data from TSQ Vantage were analyzed using Skyline23

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software to produce the peak area of extracted ion chromatograms (XIC) from multiple

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transitions monitored for target peptides. The S/N for LC-SRM measurements were

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calculated by the peak apex intensity over the highest background noise in a retention

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time region of ± 30 seconds for each target peptide. All data were processed by the

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program automatically and were later manually inspected to ensure correct peak detection

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and accurate integration.

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Results and Discussion

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Characterization of the Orthogonal Injection HPIF. The optimal tuning of optics

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for orthogonal ion injection was expected to be different from the inline injection since

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ions experience significantly different gas dynamics. The characterization of RF

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amplitude was first performed to provide optimal radial confinement. During the

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experiment the pressures of HPIF, IFT and analyzer chambers were maintained at 8, 1

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and 3×10-6 Torr respectively. Figure 2a shows the Total Ion Current (TIC) for

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singly-charged polytyrosine-1, 3, 6 as a function of the RF amplitude with frequency of 1

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MHz in the orthogonal (orange squares) and inline injection (aqua diamonds) modes. The

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ion signal in the inline mode reached a plateau at lower RF amplitude of 120 Vp-p, in

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comparison to the orthogonal mode at 160 Vp-p, which indicates that a stronger RF

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confinement in radial direction is required for orthogonal injection. The higher RF

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confinement, presumably, is required to overcome the effect of gas dynamics in the

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orthogonal direction and prevent ions from hitting the side of the HPIF. Figure 2b shows

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the normalized selected ion chromatogram (SIC) measurements for three individual ions

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as a function of the RF amplitude in the orthogonal mode. The intensity is normalized to

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the highest value for each. The highest SICs for ions of m/z 182 (green squares), 508

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(blue circles) and 997 (red triangles) were obtained with RF amplitudes of 160, 180 and

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220 Vp-p respectively. These shifts of the curves with RF amplitude are ascribed to the

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influence of low mass cut off resulting from the different RF trapping potentials. The

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results indicate that RF >220 Vp-p is sufficient for efficient ion transmission over at least a

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range of m/z 182 to m/z 997.

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Figure 2c compares TIC measurements for the orthogonal (orange squares) and

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inline (aqua diamonds) injection modes as a function of electrical field along the HPIF

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straight section. The variation of the electrical field was implemented by changing the

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voltages applied at the repeller and the HPIF entrance (and which then dictates the

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potentials applied to the HPIF electrodes through its resistor chain). As can be observed

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in Figure 2c ions, in both injection modes, are still transmitted through the straight region

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with a negative electrical field gradient due to gas dynamic effects, but as expected fewer

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ions were transmitted in the orthogonal injection mode. For instance at a repulsive

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electric field of 30V/cm no ions were transmitted in the orthogonal injection mode while

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for the inline capillary 70% of the maximum signal (for inline injection mode) is still

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observable.

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exiting the inlet capillary, which also can transmit undesirable charged droplets and

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neutral particles. The ion currents increased with a positive electrical field gradient in

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both modes. The highest observable current was at the gradient of 2.2 V/cm for inline

These results indicate that ions were accelerated by the expansion of gas

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injection and 35.1 V/cm for orthogonal injection mode when the repeller voltage reached

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to its maximum output at 380 V due to the limitations power supply. The higher field

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strength for the optimal transmission in the orthogonal injection mode reflects the

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differences in gas dynamics for the two arrangements. Contrary to the effect of DC

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gradient observed in Figure 2c the repulsive DC field on the converging section show no

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effect (Supplementary Materials Figure S1) on ion intensities in either modes, orthogonal

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or inline, due to the reduced gas dynamics in the region far away from the inlet capillary.

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Figure 2d shows the effect of the pressure on TIC measurements in the orthogonal

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mode. The ion intensities were measured as the function of HPIF chamber pressure while

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the IFT chamber pressure was maintained at 1 Torr and the RF parameters for both

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funnels were fixed at 220 Vp-p/1 MHz for HPIF and 184 Vp-p/1.3 MHz for the IFT. The

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pressures of both chambers were adjusted by choking the flow to the mechanical pumps.

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MS results optimized at 8-12 Torr, corresponding to 95% or above the maximum

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intensity achieved in the orthogonal injection mode. Insufficient RF focusing at higher

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pressure (>12 Torr) resulted in a decrease in ion intensities in both injection modes (and

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which can be mitigated by further increasing the RF amplitude).9

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The sensitivity of the orthogonal HPIF-IFT interface was first evaluated using a

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nine peptide mixture. A label-free MS quantitation of a peptide mixture (Supplementary 12


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Materials Table S1) was performed prior to the LC-SRM experiment, and baselines were

2

established using both a commercial interface (s-lens) and an inline HPIF-IF interface on

3

the same triple quadrupole MS. During the experiment the capillary temperature was set

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at 140 ⁰C for HPIF and 310 ⁰C for commercial interface (Figure 1a) and the resolution

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setting on triple quadrupole is with peak width of 0.7 Da for Q1 and Q3. The sensitivities

6

of MS quantitation and LC-SRM were assessed based on the LOD and limit of

7

quantification (LOQ) values. Figure 3 shows three randomly selected examples of

8

calibration curves from Kemptide (Figure 3a and 3b), Renin (Figure 3c and 3d) and

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Fibrinopeptide A (Figure 3e and 3f) by the orthogonal (red squares), inline HPIF-IF

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(green circles) and commercial standard s-lens interface (blue triangles). The results in

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Figure 3a, c and e show that the 4- to 32-fold improvements in LODs and overall higher

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S/N was obtained by the ion funnel configurations (orthogonal and inline) in comparison

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to the standard source. In Figure 3 b, d and f the results of intensity measurement indicate

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that the best LODs with good linearity were obtained by the orthogonal injection in

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comparison to the inline injection mode. The detailed measurement of all nine target

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peptides in Supplementary Materials Table S2 shows that orthogonal injection HPIF

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significantly improves S/N and coefficient of variation (CV) for the analytes less than

18

6.25 nmol levels. Overall the LOD and LOQ values obtained from each surrogate peptide 13



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by orthogonal injection show sensitivities 2- to 8-fold better than inline injection HPIF

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and 8- to 32-fold better than the commercial s-lens interface.

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A further evaluation on the utility of the orthogonal injection was conducted using a

4

label-free LC-SRM analysis of 18 peptides (Supplementary Materials Table S3) from

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four proteins prepared at equal concentrations ranging from 31.2 amol/µL to 2 fmol/µL

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for the three interfaces. Figure 4 illustrates the calibration curves of selected 6 target

7

peptides out of the 18 peptides analyzed. The scan width for the QqQ was set at 0.002

8

m/z and 15 ms for the scan time in the SRM mode. The sensitivities of LC-SRM data

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were assessed based on the LOD and the linearity of the calibration curves. The

10

integrated peak areas of three monitored transitions for each peptide were recorded as a

11

function of the amount loaded

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detectable concentration from orthogonal injection HPIF are estimated to be 2-fold better

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than using inline injection HPIF and 2- to 8-fold better than the s-lens interface for most

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of the peptides. The summary of quantitative performance of the 18 peptides using three

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interfaces is shown in Supplementary Materials Table S4. The better detection limit was

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obtained in 13 out of 18 peptides by orthogonal, 6 by inline injection HPIF and 3 from

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standard interface. For linearity, moderate improvements were shown using orthogonal

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injection HPIF upon the LC-SRM which also attributed to reduced chemical background.

at the LC column. The results indicate that the lowest

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1 2

Conclusions

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An orthogonal injection ion funnel interface has been designed, implemented,

4

characterized and evaluated in conjunction with a LC-SRM triple-quadrupole MS.

5

Orthogonal extraction of ions from the source gas flow path is shown to significantly

6

enhance the sensitivity for detection of analytes from peptide mixture and complex

7

biological samples. In comparison with the standard triple-quadrupole interface, the

8

significant improvement on S/N measurement and 4- to 32-fold enhancement of LOD for

9

single stage MS quantitation were achieved using a peptide mixture. For the LC-SRM

10

based protein quantification, the XICs of monitored peptides showed 2- to 8-fold

11

enhancement of the LOD with good linearity of measurements.

12 13

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Acknowledgments

2

The authors thank Dr. Tujin Shi, Dr. Ian Webb, Dr. Jonathan Cox and Dr. Jia Guo for

3

helpful discussions. The authors also thank Mr. Grant Fujimoto for the assistance for the

4

data processing programming. Portions of this research were supported by the NIH

5

National Cancer Institute (1R33CA155252) and General Medical Sciences Proteomics

6

Research Resource at Pacific Northwest National Laboratory (PNNL) (GM103493-12),

7

the Laboratory Directed Research and Development Program at PNNL, and the

8

Department of Energy Office of Biological and Environmental Research Genome

9

Sciences Program under the Pan-omics program. All the experiments were performed in

10

the Environmental Molecular Sciences Laboratory, a US Department of Energy (DOE)

11

national scientific user facility located at PNNL in Richland, Washington. PNNL is a

12

multiprogramming national laboratory operated by Battelle for the DOE under contract

13

DE-AC05- 76RLO01830.

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Supporting Information Available This information is available free of charge via the Internet at http://pubs.acs.org/

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(1) Cox, J.; Marginean, I.; Smith, R.; Tang, K. Journal of The American Society for Mass

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the American Society for Mass Spectrometry 2006, 17, 1299-1305. (10) Kim, T.; Udseth, H. R.; Smith, R. D. Analytical Chemistry 2000, 72, 5014-5019. (11) Kim, T.; Tang, K.; Udseth, H. R.; Smith, R. D. Analytical Chemistry 2001, 73, 4162-4170. (12) Whitehouse, C. M.; Welkie, D. G.; U.S. Patent US8507850 B2, 2013. (13) Tang, K.; Tolmachev, A. V.; Nikolaev, E.; Zhang, R.; Belov, M. E.; Udseth, H. R.;

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Smith, R. D. Analytical Chemistry 2002, 74, 5431-5437. (14) Mordehai, A.; Werlich, M. H. U.S. Patent 8324565 B2, 2009. (15) Senko, M. W.; Kovtoun, V. V. U.S. Patent 7514673 B2, 2013. (16) Wouters, E. R.; Splendore, M.; Mullen, C.; Schwartz, J. C.; Senko, M. W.; Dunyach, J. J. In Presented at the 57th Conference of the American Society for Mass Spectrometry: Philadelphia, PA, 2009. (17) Giles, K. U.S. Patent 12/679,139, 2011. (18) Giles, K.; Wildgoose, J.; Williams, J.; Cecco, M. D. In Presented at the 61th Conference of the American Society for Mass Spectrometry: Minneapolis, Minnesota, 2013. (19) Fialkov, A. B.; Steiner, U.; Jones, L.; Amirav, A. International Journal of Mass

34 35

Spectrometry 2006, 251, 47-58. (20) Belov, M. E.; Prasad, S.; Prior, D. C.; Danielson, W. F.; Weitz, K.; Ibrahim, Y. M.;

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Smith, R. D. Analytical Chemistry 2011, 83, 2162-2171. 18


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1

(21) Ibrahim, Y.; Belov, M. E.; Tolmachev, A. V.; Prior, D. C.; Smith, R. D. Analytical

2 3

Chemistry 2007, 79, 7845-7852. (22) Kelly, R. T.; Page, J. S.; Luo, Q.; Moore, R. J.; Orton, D. J.; Tang, K.; Smith, R. D.

4 5

Analytical Chemistry 2006, 78, 7796-7801. (23) MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen,

6 7

B.; Kern, R.; Tabb, D. L.; Liebler, D. C.; MacCoss, M. J. Bioinformatics 2010, 26, 966-968.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Repeller HPIF

(a)

IFT

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Triple Quadrupole MS Q1

Q0

Q2 1.5 mtorr

Inline Orthogonal

8 torr

4x10-6 torr

1.1 torr Q0

s-Lens

4x10-6 torr

Q3

Detector

1

(b)

2 3 4 5 6 7 8

Figure 1. (a) Schematic diagram of the triple quadrupole MS with the standard source (s-lens), orthogonal and inline high pressure ion funnel and ion funnel trap, and the ion optical configuration for the each segment along HPIF and IFT. (b) Section view of the orthogonal injection HPIF-IFT atmospheric pressure interface. Inlet capillaries of 1 mm i.d. are used for in-line and orthogonal injection.

9 10 11

20


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(b) 3x10

8

2x10

8

100

Intensity (%)

Intensity

(a)

1x108 Orthogonal Inline

0 100

200

80 60 RF frequency: 1 MHz

40

m/z 182 m/z 508 m/z 997

20 100

300

(c)

200

300

RF Amplitude (VPP)

RF Amplitude (VPP)

(d)

3x108

95%

Intensity (%)

100

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1x108

Orthogonal Inline

1 2 3 4 5 6 7 8

-40

-20

0

20

60 40 20

0 -60

80

8

40

10

12

14

16

18

Pressure (Torr)

E (V/cm)

Figure 2. (a) Comparison of total ion current as the function of RF amplitude in orthogonal and inline injection modes. (b) Comparison of ion intensities from ions of m/z 182, 508 and 997 as the function of RF amplitude varied from 60 to 300 Vp-p in orthogonal injection mode. (c) Measurement of ion transmission efficiency at HPIF straight section, and (d) Effect of pressure on ion transmission for the HPIF in orthogonal and inline injection mode.

21



(b)

200

Kemptide Orthogonal Inline sLens

100

0

(c)

0

100

Kemptide 2+, m/z 387 1x107

1x108

(d)

0 0

0

25

50

0

6x10

200

Renin 3+, m/z 587

3x106

3x107

(f) Fibrinopeptide

0 0

0

50

400

7 5x106

(e) 120

25

Orthogonal Inline

Renin

30

0

8

0

Intensity

s/n

60

50

2x10

3x107

Intensity

s/n

(a)

5x10

12

24

0

200

400

7

Fibrinopeptide A 2+, m/z 769

60

0

1 2 3 4 5

Intensity

3x106

s/n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

0

50

100

2x106

3x107

0 0

0

12

24

0

Concentration (nmol/µL)

200

400

Concentration (nmol/µL)

Figure 3. Three selected calibration curves from the 9 peptide mixture for concentrations ranging from 200 pmol/µL to 400 nmol/µL using orthogonal injection HPIF, inline injection HPIF and standard triple quadrupole atmospheric pressure interfaces.

22


Page 23 of 24

EDLIAYLK

Intensity (a.u.)

3.5x108

1.8x108

0.0

2.5x108

0.5

Intensity (a.u.)

1.0

1.5

0.0

2.0

IVGGWECEK

0.0

1.0

1.5

2.0

LFTGHPETLEK

1.8x108

0.5

1.0

1.5

0.0

2.0

LWSAEIPNLYR

0.5

1.0

1.5

2.0

TGPNLHGLFGR

1.2x108

1.8x108

0.0

0.5

3.5x108

1.5x108

3.5x108

6.0x107

0.5

1.0

1.5

0.0

2.0

Concentration (fmol/µL)

0.5

1.0

1.5

2.0

Concentration (fmol/µL)

1 2 3 4 5 6

HGFLPR

5.0x108

Orthogonal Inline sLens

3.0x108

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Typical calibration curves for 18 peptide mixed solution at concentration from 1.56 amol/µL to 2 fmol/µL using LC through orthogonal injection HPIF, inline injection HPIF and standard triple quadrupole atmospheric pressure interfaces to MS in SRM mode.

23



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Table of Contents Graphics

2

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Orthogonal Injection Ion Funnel Interface Providing ... - ACS Publications

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