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Increasing Molecular Coverage in Complex Biological &

Environmental Samples Using Ion Mobility-Mass Spectrometry

Erin Shammel Baker Xueyun Zheng, Kristin E. Burnum-Johnson, Yehia M. Ibrahim, Jennifer E. Kyle, Daniel J. Orton and Richard D. Smith Pacific Northwest National Laboratory

Multi-omic Future

MS-based Measurements

For Research Use Only. Not for use in diagnostic procedures.

p (Drift Gas)

Ffriction

Ion

Fel

Ion mobility concept

velocity is constant

v = K E

K = ion mobility

Drift Cell

E


E

in out

Drift Time

Pulse of 2 ions with

same m/z but different

shape

Different conformers separate

in time with peak heights representing

the amount of each


velocity is constant

v = K E

K = ion mobility

Drift Cell


LC (minutes) IMS (~60 ms) MS (~100 µs)

IMS MS

Elution Time Drift Time m/z

0 10 20 30 40 50 60

Elution Time (minutes)

Inte

nsi

ty

20 30 40 50 60

Drift Time (ms)

20 30 40 50 60

Drift Time (ms) 20 30 40 50 60

Drift Time (ms)

1100

100

m/z

1100

100

m/z

1100

100

m/z



How can IMS help with multi-omic analyses?

1. IMS is fast (millisecond time scale) and can be easily coupled with front-end

separations and MS

2. IMS provides a shape separation able to distinguish and assess isomers potentially

related to disease states (structural biomarkers)

3. Multi-dimensional LC-IMS-MS analyses have lower false discovery rates than LC-MS

alone

4. Fast or no LC separations can be used with IMS-MS while still detecting many features

Introduction


Spiking

Level Non-Serum Peptide

60-min LC-

IMS-TOF MS

60-min LC-

TOF MS

100-min LC-

Velos-Orbitrap

100 pg/mL Melittin ND ND ND

100 pg/mL Dynorphin A Porcine Fragment 1-13 ND ND

1 ng/mL Des Pro Ala Bradykinin ND ND

1 ng/mL Leucine Enkephalin ND ND

10 ng/mL 3X FLAG Peptide ND

10 ng/mL Substance P

100 ng/mL [Ala92]-Peptide 6

100 ng/mL Methionine Enkephalin

8 peptides spiked in human serum

Sample analyzed using Velos-Orbitrap, TOF MS and IMS-TOF MS instruments

E. S. Baker, et al. JPR, 2010.


Benefit of IMS drift time separation Improved Sensitivity & Increased Feature Detection

IMS-QTOF MS of Bradykinin (100 pM) QTOF MS of Bradykinin (100 pM)


Applications of IMS-MS to Protein Studies


60 patients

30 samples

14 samples

16 samples

Chronic liver disease from Hepatitis C

Discovery Phase: 60 matched (age, sex, fibrosis stage) samples

Correlated by biostatistician

E. S. Baker, et al. MCP, 2014.

• Estimated 130 millions people affected worldwide • No vaccine


Liver fibrosis study Discovery Phase

• Analyzed 60 post-liver transplant samples with LC-IMS-MS

• Statistical analysis identified 136 proteins that distinguish between conditions

• At least 2 unique peptides were required to identify a protein

• Significant peptides have p and q values <0.05

Non- Progressors Non- Progressors Slow Progressors Fast Progressors

Decrease = Green Increase = Red

Pro

tein

s

Pro

tein

s


Liver fibrosis study Non-transplant Comparison

• Analyzed 60 non-transplant samples with Ishak score 0-1 versus 4-6

• 63 statistically significant proteins between conditions

Non-Transplant 78

Significant Proteins

Transplant 136 63


Liver fibrosis study


Challenges in proteomic studies

1. Tissue and plasma samples are usually fractionated to detect the highest number of proteins

2. Fractionation increases analysis time, introduces errors, and causes

quantitation difficulties (peptides split between fractions)

3. Targeted approaches focus only on peptides in the target list

4. Discovery approaches do not have the sensitivity of targeted analyses

Plasma Sample Fractionation


Proteomic targeted approaches

K. E. Burnum-Johnson, et al. MCP, 2016.

Selected Reaction Monitoring

Parallel Reaction Monitoring


Can we combine discovery and targeted approaches?

K. E. Burnum-Johnson, et al. MCP, 2016.

Discovery & Targeted Monitoring (DTM)

Heavy labeled peptide

Endogenous peptide

Drift Time

m/z

583

584

585

586

587

588

589

590

591 1 0


Add heavy labeled peptides C-terminal [13C6

15N2] Lys = 8 Da m/z shift C-terminal [13C6

15N4] Arg = 10 Da m/z shift

Breast cancer derived xenograft (PDX) tissue sample

Mice engrafted bilaterally with WHIM16 breast tumor (Luminal A)

6-9 weeks

WHIM16 PDX samples for analyses

Pooled, cryopulverized and spiked with 20 heavy peptides of interest

Li et al, Cell Rep. 2013, 4: 1116-30 and Zhang et al, Nature. 2014, 513: 382-7


CPTAC_LabelFree_P6_4_17Sep14_Frodo_14... 9/17/2014 11:03:30 PM

RT: 0.00 - 99.00

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

NL: 2.38E9

TIC F: FTMS + p NSI

Full ms

[400.00-2000.00] MS

CPTAC_LabelFree_P6_

4_17Sep14_Frodo_14-

04-13

RT: 0.00 - 99.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

DTM approach


Analysis of EGFR peptide (TIQEVAGYVLIALNTVER)3+


RT: 0.00 - 99.00

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

NL: 2.38E9

TIC F: FTMS + p NSI

Full ms

[400.00-2000.00] MS

CPTAC_LabelFree_P6_

4_17Sep14_Frodo_14-

04-13

RT: 0.00 - 99.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

40 fmol/µg heavy added

Heavy labeled peptide

Endogenous peptide



RT: 0.00 - 99.00

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

NL: 2.38E9

TIC F: FTMS + p NSI

Full ms

[400.00-2000.00] MS

CPTAC_LabelFree_P6_

4_17Sep14_Frodo_14-

04-13

RT: 0.00 - 99.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

10 fmol/µg heavy added 40 fmol/µg heavy added

No heavy added

Analysis of EGFR peptide (TIQEVAGYVLIALNTVER)3+

DTM Analyses

Kynureninase peptide (IEDILEVIEK)2+, DTM = 1.49 fmol/µg

2 fmol/µg heavy

Transmembrane peptide (ALPILEELLR)2+, DTM = 16.40 fmol/µg

20 fmol/µg heavy


Targeted comparison

Good agreement between SRM and DTM


Unique Peptides with <1% FDR

QExact 17289

VOrbi 16666

IMS 22401

Comparison with discovery-based approaches


Unique Peptides with <1% FDR Non-redundant Human Proteins with at least 2 peptide identification

8,055 peptides and 319 proteins were detected only by IMS-MS

QExact 17289

VOrbi 16666

IMS 22401

QExact 2164

VOrbi 2070

IMS 2618

Comparison with discovery-based approaches


Sequence coverage difference

Of the 1855 proteins detected by all platforms, IMS-MS has the least number with only 1 or 2 ids and the highest number with more than 4 ids (best sequence coverage)

Of the 1855 proteins detected by all platforms

# of Peptides/Protein IMS QE VOrbi

1 161 245 308

2 230 317 299

3 252 259 240

>4 1212 1034 1008

Non-redundant Human Proteins


Sensitivity difference

The 8055 peptides detected only by IMS-MS had lower intensities than the peptides found in all platforms (IMS more sensitive)

9832 peptides detected by all platforms

8055 peptides detected only by IMS-MS Unique Peptides


Biological importance

297 IMS-MS unique proteins found in Human Protein Atlas–Cancer database

319 proteins only detected by IMS-MS


Biological importance

49 of these ranked as ‘High’ in at least 25% of breast cancer stains All with functional roles in the biological processes necessary for cancer progression

297 IMS-MS unique proteins found in Human Protein Atlas–Cancer database

319 proteins only detected by IMS-MS


Summary

1. Multidimensional analyses with LC, IMS and MS enabled discovery and targeted (DTM)

measurements

2. The DTM targeted measurements performed similarly to SRM

3. In the DTM discovery analyses, more peptides were detected than conventional

platforms

4. DTM can be applied to any omics approach (proteomics, metabolomics or lipidomics)


Acknowledgements • Agilent Technologies

• National Institute of Environmental Health

Sciences of the NIH (R01ES022190)

• NIH: General Medical Sciences Proteomics

Center at PNNL (2 P41 GM103493-11),

National Institute of General Medical

Sciences and National Cancer Institute

• PNNL Laboratory Directed Research and

Development Program

• Environmental Molecular Sciences

Laboratory

Biological Separations & Mass Spectrometry Group


Biological Separations & Mass Spectrometry Group

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