SUPPORTING INFORMATION
Combined use of irreversible binding and multiple reaction monitoring technology for low- and ultra-low copy-number protein detection and
quantitation.
Arthur T. Kopylov, Victor G. Zgoda, Andrew V. Lisitsa, Alexander I. Archakov
Human blood
320 ml
Human plasma
200 ml
Concentration 61.5 mg/mL (BCA assay)
Concentrating of human plasma
150 ml
Concentration 80 mg/mL (BCA assay)
Aliquotes of human plasma
10 tubes x 15 mL
Total protein amount 12 g
Depleting of human plasma on MARSTM column
2 parallel MARS (10 x 100 mm) systems
2 systems x 5 tubes x 60 runs = 300 runs
Depleted human plasma I
10 tubes x 50 mL of depleted human plasma I = 500 mL
Desalting / Concentrating on 5 KWCO spin columns
10 tubes x 4 mL of desalted depleted human plasma = 40 mL of depleted desalted concentrated
human plasma II
Dilution of human plasma II to the original volume of human
plasma I
10 tubes x 4 mL of human plasma II  10 tubes x 50 ml of human plasma III
Measurement of protein concentration in depleted desalted diluted human plasma III
10 tubes x 50 mL of depleted desalted diluted human plasma III
= 500 mL of human plasma III
Concentration 7.81 mg/mL
Supporting Information Figure 1. General scheme of preparation and depleting human plasma on Agilent
MARSTM Hu-14 multiaffinity removal system.
Supporting Information Figure 2. Workflow of determination of LLOD in presence of depleted human plasma proteins without
application of non-specific enrichment. Concentration of spiked CYP102 and BSA proteins in the dilutions is varied from 10 -9 M to 10-15
M. Total amount of on-column peptides loaded in each dilution after tryptic digestion is 10 µG in volume 10 µL
Supporting Information Figure 3. Workflow of determination of LLOD of CYP102 and BSA proteins in presence of depleted human plasma proteins
after application of non-specific enrichment on CNBr-activated sepharose 4B. Ten µL of concentrated sample were loaded onto the column to perform
calibration in range of initial concentrations of targeted proteins, CYP102 and BSA, from 10 -18 M to 10-12 M.
Supporting Information Table 1. CYP102 peptides and parameters used for MRM method development
Peptide ID
CYP102P1
CYP102P2
CYP102P3
Sequence
Charge
state
GFAPQVATLDSHAGNLPR
617.65
3+
LWPTAPAFSLYAK
732.88
2+
SPHEDYLYQEELENAQSEGIITLHTAFSR
845.16
4+
855.89
2+
875.92
2+
CYP102P4
DFAGDGLFTSWTHEK
CYP102P5
Precusor
ion m/z
QFVDWLDQASADEVK
Fragment ion
m/z
Fragment ion
type
851.454
788.419
739.894
731.381
675.863
626.328
556.323
525.948
426.231
382.714
1165.62
967.524
896.49
583.313
484.262
448.751
381.214
300.17
1005.42
994.022
937.468
932.496
718.356
581.301
567.248
888.428
724.865
514.259
506.184
413.215
391.152
334.132
276.155
962.449
847.419
719.362
659.287
561.292
490.245
472.223
357.201
Y8
Y152+
Y142+
Y14-NH32+
Y132+
Y122+
Y5
Y153+
Y82+
Y72+
Y11
Y9
Y8
Y112+
Y92+
Y82+
Y3
B2
B8
Y182+
Y172+
Y8
Y6
Y5
B92+
Y7
Y132+
Y4
B5
Y3
B4
B3
Y2
Y9
Y8
Y7
B5-NH3
Y5
Y4
B4-H2O
Y3-H2O
Collision
energy, eV
Retention time,
min
29.50
21.7
23.05
34.7
27.32
40.1
27.72
16.33
31.50
31.5
Supporting Information Table 2. BSA peptides and parameters used for MRM method development
Peptide
ID
Sequence
Precusor
ion m/z
Charge
state
BSAP2
DAFLGSFLYEYSR
784.36
2+
BSAP3
LFTFHADICTLPDTEK
925.95
2+
BSAP6
LGEYGFQNALIVR
740.39
2+
BSAP7
TVMENFVAFVDK
700.38
2+
Fragment ion
m/z
Fragment ion
type
1121.494
717.299
1234.578
1064.473
977.436
830.382
554.238
334.129
1342.631
323.6801
471.1967
814.3861
869.4127
1019.5089
795.8698
631.8197
1017.569
300.148
375.666
571.391
685.422
813.482
960.542
1180.635
571.382
500.348
1199.557
1068.517
825.435
939.474
579.3
508.267
520.248
361.195
Y9
Y5
Y10
Y8
Y7
Y6
Y4
B3
Y13
B52+
Y122+
B7-H20
Y152+
Y9
Y14
B112+
Y9
B3
A7-NH32+
Y5
Y6
Y7
Y8
Y10
Y5
Y4
Y10
Y9
Y7
Y8
Y5
Y4
B92+
Y3
Collision
energy, eV
Retention
time, min
25.01
29.5
32.38
22.1
23.33
24.3
21.81
18
Supporting Information Figure 4. Base peak MS chromatograms of CYP102 (a) and BSA (b) peptides after digestion with
trypsin. The signals were measured using a high resolution Q-TOF mass spectrometer (resolution 18200 at m/z 400, 4GHz
mode) calibrated with an error of measurement of ±0.8 ppm. The peptides used for further analysis are indicated with arrows.
Supporting Information Figure 5. Q-TOF MS/MS spectra of the precusor ions m/z 617.653+ (a) and m/z 875.922+ (c) of CYP102 and
m/z 740.392+ (b) and m/z 784.362+ (d) of BSA with indicated fragment ions. The concentration of each protein made 0.05 μM and 1 μl of
the sample was subjected onto the column
Supporting Information Figure 6. MRM chromatograms of collision-induced dissociation products of CYP102 (m/z 617.65 3+675.82+ and m/z
875.922+962.51+) and BSA (m/z 740.392+685.41+ and m/z 784.362+ 1121.51+) peptide ions at 10-16 M concentration, corresponding to
detection limit of the pure protein standards.
Supporting Information Figure 7. MRM chromatograms of human plasma performed with method established for CYP102 and BSA. Most of the
plasma protein peptides with a sort of resemble transitions are eluted between 12 and 18 minutes and no peptides corresponding to BSA or
CYP102 were observed between 21 and 32 minutes.
Supporting Information Figure 8. Dependence of the peaks areas of pure standards CYP102 (a) and
BSA (b) after enrichment on CNBr-activated Sepharose 4B by non-specific irreversible binding from a
sample volume of 10 ml. Detection limit for both the proteins was determinedto be 10 -18 M of original
(before enrichment) concentration. The calibration curves plotted for transitions of two CYP102 peptides
(precursor m/z 617.653+ solid line “a”/ m/z 875.922+ dotted line “a”) and two BSA peptides (precursor m/z
740.392+ solide line “b”/ m/z 784.362+ dotted line “b”) in five replicates.
*
*
*
*
*
*
*
Supporting Information Figure 9. Scan spectra obtained on QqQ (scan range m/z 500 - 1100) of synthetic peptides mixture CYP102
(QFVDWLDQASADEVK, Mr 1750.88 Da, m/z 876.452+; GFAPQVATLDSHAGNLPR, Mr 1851.05 Da, m/z 617.023+/926.532) and BSA
(DAFLGSFLYEYSR, Mr 1567.72, m/z 784.862+; KVPQVSTPTLVEVSR, Mr 1639.91 Da, m/z 547.64+2/820.963+) recorded at direct
infusion into the ion source at concentration 10-9 M (a), 10-10M (b), 10--11 M (c), 10-12 M (d), 10-13 M (e), 10-14 M (f) and blank infusion (g).
*- The peptide KVPQVSTPTLVEVSR from bovin serum albumin was synthesized instead of the peptide LGEYGFQNALIVR with Mr 1479.7 Da. The reason is failed
to synthesize the peptide LGEYGFQNALIVR due to problems with the peptide synthesis and purification. The replaced peptide, LGEYGFQNALIVR, from BSA is
84% identical to the peptide LGEYKFQNALLVR from HSA.