Supplemental figures.

1
Supplemental figures. Observed extracted ion
chromatograms (XICs) of each of the ten peptides
from five standard proteins using dual ion funnel
and standard Thermo interface. Data were analyzed
using Thermo Xcalibur 2.0.7 with Qual Browser.
Six standard peptides, bradykinin fragment 1-7,
kemptide, melittin, methionine enkephalin, renin
substrate porcine, and D-Ala2-deltorphin II
were used as QC peptides for LC retention time
markers and for monitoring the overall
performance of the platform. The peak widths for
each peptide were determined by averaging the
data obtained using two highest spiked
concentrations (S8 and S9) and the elution time
difference observed relative to the nearest QC
marker peaks served as a peak integration window
for the lower (S0-S7) spike concentrations.
Analyte peak identification at the lower
concentrations were determined based on three
main criteria 1) difference of elution time from
nearest QC peaks within 10 of the average
difference observed from the two highest
concentrations 2) analyte peak widths were
similar (i.e., within 10) to the average of
peak widths from the two top concentrations and
3) analyte peak area decreases with decreasing
analyte concentration levels. On each of the
following page, the top and bottom panels
represent XICs from dual ion funnel interface and
standard Thermo interface experiments,
respectively. Top three spectra on each panel are
three selective transitions from a specific
peptide. One of the best transitions is selected
for LOD and reproducibility analysis for each
peptide. All of the transitions used in this
study are listed in supplemental Table 1. The
bottom spectrum is of the nearest QC peptide used
as an elution marker for peak identification. For
comparative study, representative spectra from
LOD concentration, one concentration above and
below this concentration, and highest
concentration are presented using both dual ion
funnel and standard Thermo interface. Analyte
peaks of interest identified are shaded in gray
for ease of visualization and arrows on the
extreme right hand side spectra (lower
concentration) indicate possible analyte peak
position embedded in / interfered with the noise
levels / matrix peaks limiting the detection.
2
a) Dual ion funnel interface
40 µg/mL
400 ng/mL
40 ng/mL
80 ng/mL (LOD)
b) Standard Thermo interface
80 ng/mL
40 µg/mL
4 µg/mL
400 ng/mL (LOD)
A XICs of peptide VLDALDSIK. LODs were
determined for transition of m/z 487.282 gt
874.49 . Methionine enkephalin (transition
m/z 574.162 gt 397.24) was used here as a
retention time marker.
3
a) Dual ion funnel interface
40 µg/mL
80 ng/mL
8 ng/mL
40 ng/mL (LOD)
b) Standard Thermo interface
40 ng/mL
40 µg/mL
400 ng/mL
80 ng/mL (LOD)
B XICs of peptide DFPIANGER. LODs were
determined for transition of m/z 509.752 gt
378.70 . Bradykinin fragment 1-7 (transition
m/z 379.142 gt 527.31) was used here as a
retention time marker.
4
a) Dual ion funnel interface
40 µg/mL
400 ng/mL
40 ng/mL
80 ng/mL (LOD)
b) Standard Thermo interface
80 ng/mL
40 µg/mL
4 µg/mL
400 ng/mL (LOD)
C XICs of peptide VDEDQPFPAVPK. LODs were
determined for transition of m/z 671.342 gt
755.45 . Methionine enkephalin (transition m/z
574.162 gt 397.25) was used here as a retention
time marker .
5
a) Dual ion funnel interface
40 µg/mL
400 ng/mL
4 µg/mL (LOD)
b) Standard Thermo interface
40 µg/mL
4 µg/mL (LOD)
400 ng/mL
D XICs of peptide LWSAEIPNLYR. LODs were
determined for transition of m/z 681.362 gt
662.36 . Renin substrate porcine (transition
m/z 587.343 gt 696.30) was used here as a
retention time marker.
6
a) Dual ion funnel interface
40 µg/mL
400 ng/mL
40 ng/mL
80 ng/mL (LOD)
b) Standard Thermo interface
40 µg/mL
400 ng/mL
80 ng/mL (LOD)
40 ng/mL
E XICs of peptide LFTGHPETLEK. LODs were
determined for transition of m/z 424.563 gt
506.26 . Bradykinin fragment 1-7 (transition
m/z 379.142 gt 527.31) was used here as a
retention time marker for accurate peak
identification. On dual funnel interface, an
interference between analyte peak of interest and
a matrix peak confounded quantification of
proteins at lt80 ng/mL.
7
a) Dual ion funnel interface
40 µg/mL
4 µg/mL (LOD)
400 ng/mL
b) Standard Thermo interface
40 µg/mL (LOD)
4 µg/mL
400 ng/mL
F XICs of peptide YLEFISDAIIHVLHSK. LODs were
determined for transition of m/z 629.013 gt
740.42 . Renin substrate porcine (transition
m/z 587.342 gt 696.30) was used here as a
retention time marker for accurate peak
identification.
8
a) Dual ion funnel interface
40 µg/mL (LOD)
4 µg/mL
400 ng/mL
b) Standard Thermo interface
40 µg/mL (LOD)
4 µg/mL
400 ng/mL
G XICs of peptide HIATNAVLFFGR. LODs were
determined for transition of m/z 673.372 gt
1095.59 . D-Ala-2-Deltorphin II (transition
m/z 783.192 gt 709.22) was used here as a
retention time marker.
9
a) Dual ion funnel interface
40 µg/mL
400 ng/mL
40 ng/mL
80 ng/mL (LOD)
b) Standard Thermo interface
40 µg/mL
4 µg/mL (LOD)
400 ng/mL
H XICs of peptide GGLEPINFQTAADQAR. LODs were
determined for transition of m/z 844.422 gt
666.34 . Methionine enkephalin (transition m/z
574.162 gt 397.25) was used here as a retention
time marker .
10
a) Dual ion funnel interface
40 µg/mL
400 ng/mL
40 ng/mL
80 ng/mL (LOD)
b) Standard Thermo interface
80 ng/mL
40 µg/mL
4 µg/mL
400 ng/mL (LOD)
I XICs of peptide EDLIAYLK. LODs were determined
for transition of m/z 482.772 gt 494.30 .
D-Ala-2-Deltorphin II (transition m/z
783.192 gt 709.22) was used here as a retention
time marker.
11
a) Dual ion funnel interface
40 µg/mL
80 ng/mL
8 ng/mL
40 ng/mL (LOD)
b) Standard Thermo interface
80 ng/mL
40 µg/mL
4 µg/mL
400 ng/mL (LOD)
J XICs of peptide TGQAPGFSYTDANK. LODs were
determined for transition of m/z 728.842 gt
1099.51 . Bradykinin fragment 1-7 (transition
m/z 379.142 gt 614.36) was used here as a
retention time marker.