In-depth proteome and phosphoproteome profiling of ...
Transcript of In-depth proteome and phosphoproteome profiling of ...
In-depth proteome and phosphoproteome profiling of nanoscale sample by data-independent acquisition mass spectrometry
Yi-Ting Wang1, Chia-Feng Tsai1, Rosalie K. Chu1, Guo Ci Teo2, Ronald J. Moore1, Hui Zhang3, Alexey I. Nesvizhskii2, Karin D. Rodland1, Richard D. Smith1, Tujin Shi1, and Tao Liu1
1Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 2Department of Pathology, University of Michigan, Ann Arbor, MI, 3Department of Pathology, Johns Hopkins University, Baltimore, MD
[1]. Dou et al. Anal Chem. 2019, 91, 9707-15. [2]. Neumann et
al. Nature Biotech. 2012, 30, 918-20. [3] Kong et al. Nature
Meth. 2017, 14, 513-20. [4] Demichev et al. Nature Meth. 2020,
17, 41-4. [5] Tsou et al. Nature Meth. 2015, 12, 258-64. [6] Tsai
et al. Mol Cell Proteomics. 2020, 19, 828-38.
www.omics.pnl.govCONTACT: Yi-Ting Wang, Ph.D.
1. Bulk proteome analysis
2. DDA vs. DIA for nanoscale samples
Pro
teo
me
Ph
osp
ho
pro
teo
me
❑ 1 ng
❑ 10 ng
❑ 1 mg
❑ 10 mg
Figure 2. The comparison of number of peptide/protein identifications from
0.5 mg AML peptides (a). The Pearson correlation of XIC area (b), PCA (c)
and CV distribution (d) were generated for the Library DIA analyses.
Figure 3. The comparison of numbers of peptide/protein (a) and
phosphopeptide (b) identifications using the DDA and DIA methods.
3. Mimic single-cell proteome analysis
Figure 4. Mimic single-cell proteome analysis using DIA. The numbers of peptides (a) and
proteins (b) were identified by direct and library DIA for 0.1 ng (single-cell), 10 ng and 100 ng
peptides. The LFQ quantitation results by library DIA were shown for the different input
levels (c).
Results
Fractions
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 F24
No. of pe
ptide
s
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
Fractions
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 F24
No
. of pro
tein
s
0
1000
2000
3000
4000
5000
6000
Proteome
F1
20 F2
7 16 F3
7 8 20 F4
8 7 11 22 F5
11 10 12 16 28 F6
Phosphoproteome
F1 F2 F3 F4 F5 F6
No
. o
f p
ho
sp
ho
pe
ptid
es
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
Tip-IM
AC
nan
oFA
C[1
]
Phosphoproteome
500mg
Digestion
Proteome
50mg
Inte
grat
ed IM
AC
-H
pH
-Tip
Single-shot DIA
Spectrum library
DIA
an
aly
sis
Figure 1. The DIA workflow. For spectrum library generation, the global peptides were fractionated by nanoflow fractionation with
automated concatenation (nanoFAC)1 while phosphopeptides were enriched and fractionated using an integrated Tip-IMAC and
Tip-high-pH fractionation, both followed by DDA LC-MS/MS analysis. The spectrum library was then generated by MSFragger and
imported into DIA-NN. Single-shot DIA data were analyzed using DIA-NN (Library DIA) or DIA UMPIRE (direct DIA).
Sp
ec
tru
m l
ibra
ry g
en
era
tio
n
% of overlap
163,002 peptides; 9,969 proteins44,273 phosphopeptides
Mixed AML cells
Mixed AML cells
Library DIA
DIA-NN[4]
Direct DIA
[5]
[3]
[2]
Methods
(a) (b) (c)
(d)
(c)(b)(a)
Portions of the research were supported by a U24CA210955 grant (to TL and RDS) and U01CA214116 grant (to KDR) from the NCI Clinical Proteomic Tumor AnalysisConsortium (CPTAC), and a P41 GM103493 grant from NIGMS (to RDS). This work was performed at the Environmental Molecular Sciences Laboratory (grid.436923.9), a U.S.Department of Energy National Scientific User Facility located at the Pacific Northwest National Laboratory operated under contract DE-AC05-76RL01830.
References: Acknowledgments:
Conclusions
1. Library generation: From small-sized samples, comprehensive
library can be created for library DIA analysis using either the recently
developed nanoFAC system or an integrated Tip-based IMAC and
manual high-pH RPLC fractionation approach for proteome and
phosphoproteome analysis, respectively.
2. Bulk proteomics: Over 6,500 protein could be detected in single-
shot DIA analysis of AML cells, with excellent reproducibility
(CV<11%).
3. Nanoscale phosphoproteomics: DIA with variable sequential
window could identify over 7,700 and 16,000 phosphopeptides from
only 1 mg and 10 mg tryptic digest, respectively.
4. Single-cell proteomics: The DIA methods provide higher detection
sensitivity than DDA methods (where up to ~2,000 proteins were
detected with isobaric labeling-based boosting and increased ion
injection times[6]). Over 9,500 peptides and 2,800 protein can be
detected by library DIA at single-cell level.
Tryptic digest