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.

yi-ting.wang@pnnl.gov

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