of 9
S
1
Supporting Information
Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single
-
Cell Equivalent
Samples
Marion Pang
1, *
,
Jeff
J Jones
2
, Ting
-
Yu Wang
1,3
, Baiyi Quan
1,3
, Nicole J Kubat
2
, Yanping Qiu
1,3
, Michael L
Roukes
1,2,*
, Tsui
-
Fen Chou
1,3,*
1
Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd.,
Pasadena, CA 91125
2
Division of Physics, Mathematics and Astronomy, California Institute of Technology, 1200 E. California
Blvd., Pasadena, CA 91125
3
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, 1200 E.
California Blvd., Pasadena, CA 91125
*Correspondence to:
marion.pang@caltech.edu
,
roukes@caltech.edu
,
tfchou@caltech.edu
Table of Contents
Supplemental Table S1. Gradient conditions for LC
-
MS/MS analysis
................................
.........................
S
2
Supplemental Table S2. MS settings for DIA LC
-
MS/MS analysis
................................
................................
S
3
Supplemental Figure S1. Further characterization of proteomic data from different proteases
...............
S
4
Supplemental Figure S2. LCMS experimental details from different proteases
................................
.........
S
4
Supplemental Figure S3.
Proteomic
DIA data from different proteases
at single
-
cell equivalent
..............
S
4
Supplemental Figure S4. Further characterization extraction and identification of peptide features across
various acquisition times
................................
................................
................................
............................
S
5
Supplemental Figure S5. LCMS experimental details
from different proteases (LysC, trypsin) across
various acquisition times
................................
................................
................................
............................
S
6
Supplemental Figure S6. CV distributions of shared and unique peptides across various acquisition times
................................
................................
................................
................................
................................
....
S
7
Supplemental Figure S7. Further characterization of proteomic
s
data from
bulk digest and dil
-
dig Tryp
and LysC
................................
................................
................................
................................
......................
S
7
Supplemental Figure S8.
CV distribution for peptides that shared and unique peptides across bulk digest
and dilute
-
then
-
digest methods at single
-
cell equivalent
................................
................................
..........
S
8
Supplemental Figure S9. LCMS experimental details from bulk digest and dilute
-
then
-
digest methods
across various input loads at digestion
................................
................................
................................
.......
S
9
S
2
Supplemental Table
S1. Gradient conditions for LC
-
MS/MS analysis
50 min gradient
Time (min)
Duration (min
)
F
low rate
(
l/min)
%
Solvent A
%
Solvent B
0
.0
0.0
0.220
97.0
3.0
1
.0
1.0
0.220
97.0
3.0
31
.0
30.0
0.220
81.0
19.0
41
.0
10.0
0.220
71.0
29.0
51
.0
10.0
0.220
59.0
41.0
52
.0
1.0
0.220
5.00
95.0
52
.0
Column Wash
57
.0
5.0
0.220
5.0
95.0
57
.0
Stop Run
57
.0
Column
Equilibration
30 min gradient
Time (min)
Duration (min
)
Flow rate
(
l/min)
%
Solvent A
%
Solvent B
0.0
0.0
0.220
99.0
1.0
0.5
0.5
0.220
99.0
1.0
1.0
0.5
0.220
92.0
8.0
24.0
15.0
0.220
80.0
20.0
30.0
4.0
0.220
65.0
35.0
30.0
Column Wash
34.0
4.0
0.220
1.0
99.0
34.0
Stop Run
34.0
Column
Equilibration
20 min gradient
Time (min)
Duration (min
)
Flow rate
(
l/min)
%
Solvent A
%
Solvent B
0.0
0.0
0.220
99.0
1.0
0.5
0.5
0.220
99.0
1.0
1.0
0.5
0.220
92.0
8.0
16.0
15.0
0.220
80.0
20.0
20.0
4.0
0.220
65.0
35.0
20.0
Column Wash
24.0
4.0
0.220
1.0
99.0
24.0
Stop Run
24.0
Column
Equilibration
S
3
14 min gradient
Time (min)
Duration (min
)
Flow rate
(
l/min)
%
Solvent A
%
Solvent B
0.0
0.0
0.220
99.0
1.0
0.5
0.5
0.220
99.0
1.0
1.0
0.5
0.220
92.0
8.0
11.2
1.02
0.220
80.0
20.0
14.0
2.8
0.220
65.0
35.0
14.0
Column Wash
18.0
4.0
0.220
1.0
99.0
18.0
Stop Run
18.0
Column
Equilibration
10 min gradient
Time (min)
Duration (min
)
Flow rate
(
l/min)
%
Solvent A
%
Solvent B
0.0
0.0
0.220
99.0
1.0
0.5
0.5
0.220
99.0
1.0
1.0
0.5
0.220
92.0
8.0
8.0
7.0
0.220
80.0
20.0
10.0
2.0
0.220
65.0
35.0
10.0
Column Wash
14.0
4.0
0.220
1.0
99.0
14.0
Stop Run
14.0
Column
Equilibration
Supplemental Table S
2
.
MS settings
for
DIA
LC
-
MS/MS analysis
Settings
Full Scan
DIA (MS2)
Resolution
120000
60000
AGC target
(%)
300
75
Maximum injection time (s)
Auto
118ms
Scan range
375
-
1200
See below
HCD collision energy (
%)
30
Precursor mass range (m/z)
Isolation window (m/z)
Number of scan events
375
-
500
25
4
500
-
600
13
7
600
-
800
8
31
850
-
900
13
3
900
-
1200
25
9
S
4
Supplemental Figure S1. Further characterization of proteomic data from different proteases (GluC,
Trypsin, LysC). A. Proportion of proteins per abundance across dilution series. B. Plot of CV distributions
for the three enzymes at a single cell load.
Supplemental Figure S2. LCMS experimental details from different proteases
(GluC, LysC, trypsin). A. Full
-
width half maximum (FWHM) in seconds. B. Points per peak. C. Peptide Probability and D.
Chromatographic peak capacity.
Supplemental Figure S
3
.
DIA data
from different proteases
(LysC, trypsin). A. Number of protein groups.
B. Number of peptide precursors identified.
S
5
Supplemental
Figure S
4
. Further characterization
extraction and identification of peptide features across
various acquisition times
of
A.
50 min, B. 30 min, C. 20 min, D. 14 min and E. 10 min
.
S
6
Supplemental Figure S
5
. LCMS experimental details from different proteases (LysC, trypsin) across
various acquisition times
of 10min, 14min, 20min, 30min and 50min. A. Full
-
width half maximum
(FWHM) in seconds. B. Points per peak. C. Peptide Probability and D. Chromatographic peak capacity.
S
7
Supplemental Figure S
6
.
CV distributions of shared and unique peptides across various acquisition times
of 10 min
, 14 min, 20 min, 30 min and 50 min
.
Supplemental
Figure S
7
. Further characterization of proteomic data from Tryp and LysC, comparing the
dilute
-
then
-
digest (lighter color) and bulk digest (darker color) methods
. A. Number of peptide groups
across dilution series. B. Proportion of quantifiable peptides across dilution series.
S
8
Supplemental Figure S
8
. CV distribution for peptides that
shared
or are unique between LysC and
trypsin
for bulk digest (left) and dilute
-
then
-
digest (right) methods at 200pg level
.
S
9
Supplemental Figure S
9
. LCMS experimental details from bulk digest and dilute
-
then
-
digest methods
across various input loads at digestion
. A. Full
-
width half maximum (FWHM) in seconds. B. Points per
peak. C. Peptide Probability and D. Chromatographic peak capacity.