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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
For Research Use Only. Not for use in diagnostic procedures.
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
Ion mobility concept
velocity is constant
v = K E
K = ion mobility
Drift Cell
For Research Use Only. Not for use in diagnostic procedures.
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
Ion mobility concept
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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.
For Research Use Only. Not for use in diagnostic procedures.
Benefit of IMS drift time separation Improved Sensitivity & Increased Feature Detection
IMS-QTOF MS of Bradykinin (100 pM) QTOF MS of Bradykinin (100 pM)
For Research Use Only. Not for use in diagnostic procedures.
Applications of IMS-MS to Protein Studies
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
Liver fibrosis study
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
Proteomic targeted approaches
K. E. Burnum-Johnson, et al. MCP, 2016.
Selected Reaction Monitoring
Parallel Reaction Monitoring
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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
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100
Rel
ative
Abu
ndan
ce
DTM approach
For Research Use Only. Not for use in diagnostic procedures.
Analysis of EGFR peptide (TIQEVAGYVLIALNTVER)3+
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
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90
100
Rel
ative
Abu
ndan
ce
40 fmol/µg heavy added
Heavy labeled peptide
Endogenous peptide
For Research Use Only. Not for use in diagnostic procedures.
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
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
For Research Use Only. Not for use in diagnostic procedures.
Targeted comparison
Good agreement between SRM and DTM
For Research Use Only. Not for use in diagnostic procedures.
Unique Peptides with <1% FDR
QExact 17289
VOrbi 16666
IMS 22401
Comparison with discovery-based approaches
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
Biological importance
297 IMS-MS unique proteins found in Human Protein Atlas–Cancer database
319 proteins only detected by IMS-MS
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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)
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
Biological Separations & Mass Spectrometry Group