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INTRODUCTION

SILAC experiments employ metabolic incorporation of isotopically labeled-amino acids into proteins for LC-MS based quantitative proteomics studies and have predominantly used MS data dependent acquisition (DDA) strategies to date. Due to the multiplexed nature of a SILAC experiment and the accompanying increase in sample complexity, co-fragmentation of multiple precursor ions challenges the specificity of the assay and thereby the qualitative and quantitative outcome. High resolution data independent acquisition (DIA) methods, especially using in-line ion mobility separation, have the potential to overcome a number of acquisition related issues in these additionally complex samples. An informatics workflow is presented for the analysis of data independent LC-MS SILAC data sets, evaluating concurrently the overall

performance of the workflow.

A QUALITATIVE AND QUANTITATIVE ION MOBILITY ENABLED DATA INDEPENDENT SILAC WORKFLOW

Andrew JK Williamson1, Steven Ciavarini2, Scott J Geromanos2, Andrew Tudor3, Barry Dyson3, Lee Gethings3, Kelly McMahon3, Robert Tonge3, James I Langridge3, Anthony D Whetton1, Johannes PC Vissers3 1 School of Cancer and Imaging Sciences, University of Manchester, UK, 2 Waters Corporation, Milford, MA, 3 Waters Corporation, Manchester, UK

References

1. Cadeco et al. The use of proteomics for systematic analysis of normal and transformed hematopoietic stem cells. Curr Pharm Des. 2012;18(13):1730-50.

2. Richardson K et al., A probabilistic framework for peptide and protein quantification from data-dependent and data-independent LC-MS proteomics experiments. OMICS. 2012;16(9):468-82

3. Silva J et al. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Mol Cell Proteomics. 2006;5(1):144-56.

4. Martin BR et al. Global profiling of dynamic protein palmitoylation. Nat Methods. 2011 Nov 6;9(1):84-9.

Acknowledgement

Brent Martin is kindly acknowledged for providing mouse T-cell hybridoma cell data.

Figure 1. SILAC LC-IM-DIA-MS workflow.

Figure 2. Principle LC-IM-DIA-MS acquisition.

RESULTS

Proof of principle

A sample with known relative amounts was analyzed to dem-onstrate proof-of-principle of DIA and IM-DIA for SILAC analy-

sis. The workflow is shown in Figure 1, combining a tryptic di-gest of non-labeled and heavy isotope-labeled forms of sample

1, followed by LC-DIA-MS (LC-MSE) or LC-IM-DIA-MS (HDMSE), with 100 ng of material loaded in total on-column.

An identification example is shown in Figures 1 and 3. The bot-

tom right pane of Figure 1 illustrates a SILAC pair, displaying characteristic mass shift due to isotopic incorporation, a ratio

of ~ 1:2 and chromatographically eluting less than 0.02 min

apart. The two middle panes of Figure 3 show fragment ion IM-DIA spectra with the same mass shifts between the fragment

ions, providing an additional level of validation for quantifica-tion. The outer panes show part product ion spectra.

LC-MS conditions

Nanoscale LC separation of tryptic peptides was conducted

with a trap column configuration using a nanoAcquity system and a 90 min gradient from 5-40% ACN (0.1% FA) at 300 nL/

min using a BEH 1.7 µm C18 reversed phase 75 µm x 20 cm nanoscale LC column.

MS data were acquired in data independent analysis mode

(LC-DIA-MS) using a Xevo G2-S QTof mass spectrometer or in data independent ion mobility analysis mode (LC-IM-DIA-MS)

using a Synapt G2-S instrument.

Informatics

The LC-MS peptide data were processed, searched and quanti-

fied [2] with ProteinLynx GlobalSERVER v3.0 and reviewed UniProt protein sequence databases throughout.

METHODS

Sample preparation

Three SILAC sample types were investigated following reduction, alkylation and trypsin digestion:

1. Ba/F3 mouse cells comprising a Jak2-V617F mutation were

grown on 12C6- or 13C6-lysine media [1]. 2. Non-labeled UPS2 dynamic range standard was spiked into

13C6-15N4-argine 13C6-

15N2-lysine labeled HEK-293 cells. 3. Non-labeled and 13C6-

15N4-argine 13C6-15N2-lysine labeled

BW5147-derived mouse T-cell hybridoma cells.

Figure 4. Protein quantification results DIA SILAC quantitation

Ba/F3 mouse cells with median and average values of 0.47 and 0.48. Shown inset are peptide intensities and ratios for Profilin-

1 (–0.92 ± 0.11), identified on the basis of 10 peptides with precursor and product ion RMS errors of 3.2 and 4.2 ppm.

Figure 3. Example SILAC LC-IM-DIA-MS data of paired MS2

product ion spectra.

CONCLUSIONS

Data-independent (DIA) MS acquisition strategies,

incorporating high peak capacity ion mobility

separations can be routinely applied for SILAC based quantitation studies

SILAC-labeled and unlabelled peptide pairs can be

detected with high specificity and database searched

to identify proteins

Quantification measurements are accurate and

precise, taking advantage of both SILAC-pair precursor/fragment accurate mass and ionic mobility

Figure 8. Log ratio WT (light)/labeled (heavy) hybridoma cells

vs. 1 / log ratio hybridoma (light)/labeled WT (heavy) cells (average values and errors from 3 (fractions) x 2 (technical)

replicates; slope data = 0.560 with r = 0.935).

Figure 7. Reciprocal response readout principle, contrasting LC-IM-DIA-MS data from WT andlabeled BW5147-derived mouse T-

cell hybridoma cells and replicating the experiment with reversed labeling order. Example spectra from CKAP4_HUMAN.

Figure 6. Amount estimation (n = 3) of quantified non-labeled

UPS2 proteins in 200 ng SILAC medium (cyan, green and red circles). The boxes/whiskers represent the six UPS2 dynamic

range bins and the blue arrows the expected molar amounts.

L

I

G

H

T

H

E

A

V

Y

IMS CID TOF MS

A B

light AA heavy AA Incorporation of stable Isotopes (15N and 13C)

Combine Sample States A and B

Tryptic digestion (optimized with RapiGest)

Data Independent AcquisitionLC-DIA-MS (LC-MSE)

or LC-IM-DIA-MS (LC-HDMSE)

Identification andProtein / Peptide Quantification

ratio

Figure 5. UPS2 peptide identifications (no modifications spefi-

cied; grey = SILAC labeled background) and annotated IM-DIA spectrum one of the UPS2 protein spikes.

The average normalized estimated UPS2 protein amounts of

three technical replicates are shown in Figure 6 and super-imposed on the expected dynamic range [3]. In total, 27 UPS2

proteins were identified across 4 amount bins.

Peptides and proteins are quantified using a probabilistic

framework [2]. Figure 4 shows a protein summary and the de-tails of one of the quantified proteins. In combining unlabeled

and labeled-forms of an identical cell type, shown in Figure 4, as expected the majority of the proteins had the same fold-

change; ~ 80% of the data exhibits a fold change value be-tween 0.4 and 0.6, suggesting that two equivalents of unla-

belled cells were combined with one labeled equivalent.

Specificity and dynamic range

The specificity and quantitation dynamic range of the workflow

was explored by spiking non-labeled UPS2 dynamic range standard into 200 ng 13C6-

15N4-argine 13C6-15N2-lysine labeled

HEK-293 cells. Figure 6 illustrates the identification of the UPS2 spiked proteins without specifying any modifications, i.e.

only UPS2 proteins should be identified, illustrating high DIA acquisition and database search specificity.

frequency

Binned application note_light:application note_heavy_Ratio

Accuracy

The accuracy of the workflow was accessed by a reversed

labeling experiment [4] of which the principle and results are summarized in Figures 7 and 8, respectively, suggesting great

accuracy and precision but incomplete label incorporation.

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

0.5 5 50 500 5000 50000

amount bin

am

ou

nt

ran

ge

ALBU_HUMAN

HBA_HUMAN

LEP_HUMAN HBB_HUMAN

CAH1_HUMAN

CAH2_HUMAN

UBIQ_HUMAN

PRDX1_HUMAN

PPIA_HUMAN

NQO1_HUMAN

CATA_HUMAN

MYG_HUMAN

CYB5_HUMAN

CO5_HUMAN

EGF_HUMAN

SUMO1_HUMAN

am

ount

range

NEDD8_HUMAN

RETBP_HUMAN

ln ratio var = 0.05

A A

A

A B

B B

B

light AA heavy AA

combine fractions

tryptic digestion

LC-IM-DIA-MS (2 technical replicates/fraction)

MS1 (0.01 min) MS1 (0.01 min)

MS2 light MS2 light

MS2 heavy MS2 heavy

B B

B

BA

A A

A

light AA heavy AA

combine fractions

tryptic digestion

LC-IM-DIA-MS (2 technical replicates/fraction)

reversed labeling

heavy

CKAP4_HUMAN

complete (theoretical ) incorporation label)