Supporting Information
The proteomic analysis improved by cleavage kinetics-based
fractionation of tryptic peptides
Yanbo Pan,a, b Jiawei Mao,a, b Zhenzhen Deng,a, b Mingming Dong,a, b Yangyang Bian,a,
b
a
Mingliang Ye,*,a and Hanfa Zou*,a
Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic
Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Dalian 116023, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
Corresponding Author
*Prof. Hanfa Zou: phone, +86-411-84379610; fax, +86-411-84379620; e-mail,
hanfazou@dicp.ac.cn.
*Prof. Mingliang Ye: phone, +86-411-84379620; fax, +86-411-84379620; e-mail,
mingliang@dicp.ac.cn.
Contents:
Supplementary Experimental Procedures
Supplementary Table and Figures
Table S1 The identifications of peptides and proteins from samples 0-4 and control.
Fig. S1 The percentages of D and E around the K/R residues revealed by the
identified peptide termini (a) and the missed cleavage K/R residues on the identified
peptides (b).
Fig. S2 Abundance of proteins identified from samples 1-4 and control.
Fig. S3 The overlaps of identified peptides (A) and proteins (B) between samples 1-4
and control.
Fig. S4 The percentages of peptides with missed cleavage sites in different samples.
Fig. S5 Distributions of charges (a) and length (b) for peptides identified from
samples 1-4 and control.
Fig. S6 Venn diagrams of peptide and protein identifications from samples 1-4.
Fig. S7 Venn diagrams of the peptide (a) and protein (b) overlaps between any two
samples derived from the four digestion stages.
Fig. S8 Base peak chromatograms for RPLC-MS/MS analysis of samples 1-4.
Supplementary Experimental Procedures
Materials. Urea, tetraethylammonium bromide (TEAB), and trypsin [bovine, treated
with L-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)] were obtained from
Sigma-Aldrich (St. Louis, MO), Amiconultra-0.5 mL centrifugal filters was purchased
from Millipore (Bedford, MA), Deionized water used for all experiments was
prepared using a Mill-Q system (Millipore, Bedford, MA).
Extraction of HeLa cell proteins. The HeLa cells were grown and lysed according to
previous study.1 The cell pellets were softly homogenized in a cold lysis buffer,
sonicated for 120 s (400 W), and centrifuged at 23000g for 1 h. The supernatant
containing the total cell proteins was precipitated with five volumes of cold
acetone/ethanol/acetic acid (vol/vol/vol = 50/50/0.1) at −20 °C. Protein precipitant
was centrifuged at 15000g for 30 min. The pellet was washed separately with acetone
and 75% ethanol, then lyophilized to dryness, and stored at −80 °C.
Generation of tryptic peptides by using different trypsin concentration and
different digestion time. The acquired proteins (100 μg) from HeLa cells were
denatured in 100 μL of 8 M urea/100 mM TEAB (pH=8.0), the disulfide bond of
proteins was opened by incubating with 10 mM DTT for 1 h at 56 oC and then
alkylated by 20 mM IAA in the darkness at room temperature for 40 min. The mixture
was diluted to 500 μL with 100 mM TEAB (pH=8.0) and transfer to a centrifugal
filter with molecular weight cutoffs of 10000 Da (10 KDa), then centrifuged at
14000g for 20 min. The filtrate was collected and named as sample 0 to detected
endogenous peptides. The proteins in centrifugal filter were redissolved in 1.5 M
urea/100 mM TEAB (pH=8.0) and trypsin was added at a 2500:1 protein/protease
(wt/wt) ratio for digestion at 37 °C. After digested for 15min, it was centrifuged at
14000g for 20 min, and then the filtrate was collected and named as sample 1. The
proteins in centrifugal filter were redissolved in 1.5 M urea/100 mM TEAB (pH=8.0)
and digest for additional 4h at 37 °C, then centrifuged at 14000g for 20 min, the
filtrate was collected and named as sample 2. The proteins in centrifugal filter were
redissolved in 1.5 M urea/100 mM TEAB (pH=8.0) and additional trypsin was added
at a 50:1protein/protease (wt/wt) ratio for further digestion at 37 °C. After digested for
1h, it was centrifuged at 14000g for 20 min, and the filtrate was collected and named
as sample 3. The proteins in centrifugal filter were redissolved in 1.5 M urea/100 mM
TEAB (pH=8.0) and digest overnight at 37 °C, then centrifuged at 14000g for 20 min,
the filtrate was collected and named as sample 4.The five samples were acidized by
10% TFA and desalted with homemade C18 SPE columns. In a control trypsin digest
experiment (control), the same proteins were digested with a 50:1 protein/protease
(wt/wt) ratio at 37 °C overnight, ultrafiltered with another centrifugal filter, then
acidized by 10% TFA, and desalted with a homemade C18 SPE column. All of the
resulting peptide samples were lyophilized to dryness and then stored at -20 oC for
further usage.
Nano LC-MS/MS analysis. RP LC-MS/MS system consisted of a LTQ-orbitrap
Velos mass spectrometer (Thermo Fisher Scientific) with a nanospray source. A
capillary column was first manually pulled to a fine point as spray tip, and then
packed with C18 AQ beads (5μm, Michrom Bio Resources). 0.1% (v/v) formic acid in
water and 0.1 % (v/v) formic acid in acetonitrile were applied as the mobile phase.
The gradient elution from 5% to 35% (v/v) of the 0.1% (v/v) formic acid in
acetonitrile in 150 min was performed to elute each sample in 1D RPLC-MS/MS. All
MS and MS/MS spectra were acquired in the data dependent mode with the twenty
most intense ions fragmented by CID.
For 2D SCX-RP LC-MS/MS analysis with the LTQ-Orbitrap mass spectrometer
(Velos, Thermo Fisher Scientific), a capillary monolithic column (5 cm × 200 μm ID)
with phosphate functional groups was applied as an SCX trap column in the first
dimension. The sample loading and analysis procedures were as follows: the peptide
samples were first dissolved in 0.1% (v/v) formic acid in water, and then loaded onto
the monolith SCX trap column; the trap column was equilibrated with 0.1% (v/v)
formic acid in water for 10min. After that, it was directly connected to an RP
analytical column in tandem by a union. Then four gradient elution steps were applied
to gradually elute peptides from the SCX trap column to the RP analytical column
with ammonium acetate solution concentrations of 100, 300, 500, and 1000 mM,
respectively. After each elution step, a subsequent RPLC-MS/MS was executed in 150
min-gradient time with 0.1% (v/v) formic acid in acetonitrile from 5% to 35% (v/v), a
capillary column was first manually pulled to a fine point as spray tip, and then
packed with C18 AQ beads (3 μm, 120 Å, Michrom Bio Resources). All MS and
MS/MS spectra were acquired in the data dependent mode with the twenty most
intense ions were fragmented by CID.
Data analysis. Protein identification was performed using MaxQuant (version 1.3.0.5,
http://www.maxquant.org).2 The raw files were searched against UniProt database of
human
downloaded
from
http://www.uniprot.org
(releasedon12/11/2013),
carbamidomethylation on cysteine was set as a fixed modification, and oxidation on
methionine was set as variable modifications. Peptides were searched using fully
tryptic cleavage constraint and up to four missed cleavage sites were allowed, the
mass tolerances for the precursor ions and fragment ions were set to 7 ppm and 0.5 Da,
respectively. Peptide and protein candidates were filtered to 1% false-discovery rate
(FDR). The other settings were the same to the conventional search. Peptide
sequences were analysis by WebLogo (http://weblogo.berkeley.edu/logo.cgi). The
peptide sequences were centered at the cleavage sites K/R and extended 13 residues
(± 6 residues). The N- or C- terminal sequences that could not be extended were
excluded. The abundance of identified proteins was provided by PaxDB
(http://pax-db.org) 3.
References
1. Ye, M.; Pan, Y.; Cheng, K.; Zou, H., Protein digestion priority is independent of
protein abundances. Nature methods 2014, 11 (3), 220-222.
2. Cox, J.; Mann, M., MaxQuant enables high peptide identification rates,
individualized
p.p.b-range
mass
accuracies
and
proteome-wide
protein
quantification. Nature biotechnology 2008, 26 (12), 1367-1372.
3. Wang, M.; Weiss, M.; Simonovic, M.; Haertinger, G.; Schrimpf, S. P.; Hengartner,
M. O.; von Mering, C., PaxDb, a Database of Protein Abundance Averages
Across All Three Domains of Life. Molecular & Cellular Proteomics 2012, 11 (8),
492-500.
Supplementary Table and Figures
Table S1 The number of identified peptides and proteins from samples 0-4 and
control.
Control
Sample 0
Sample 1
Sample 2
Sample 3
Sample 4
Peptides
7010
233
6641
13070
12877
7101
Proteins
1566
169
1813
2327
2385
1644
Fig. S1 The percentages of D and E around the K/R residues revealed by the
identified peptide termini (a) and the missed cleavage K/R residues on the identified
peptides (b).
Fig. S2 Abundance of proteins identified from samples 1-4 and control. (The
abundance of identified proteins was provided by PaxDB, http://pax-db.org)
Fig. S3 The overlaps of identified peptides (A) and proteins (B) between samples 1-4
and control.
Fig. S4 The percentages of peptides with missed cleavage sites in different samples.
Fig. S5 Distributions of charges (a) and length (b) for peptides identified from
samples 1-4 and control.
Fig. S6 Venn diagrams of peptide and protein identifications from samples 1-4. The
peptides and proteins that only identified from one fractions were region 1, 3, 9, 14.
Fig. S7 Venn diagrams of the peptide (a) and protein (b) overlaps between any two
samples derived from the four digestion stages.
Fig. S8 Base peak chromatograms for RPLC-MS/MS analysis of samples 1-4.