new instruments and services - ohio state university...new, fast autotune improves sensitivity up to...
TRANSCRIPT
![Page 1: New instruments and services - Ohio State University...New, fast autotune improves sensitivity up to 5x for small molecule applications Autotune technology also helps preserve fragile](https://reader033.vdocuments.us/reader033/viewer/2022042206/5ea8356bcf3d5a0440764c61/html5/thumbnails/1.jpg)
New instruments and
services
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Resolving power up to 140,000 FWHM and Highmass accuracy <1ppm
Fast scanning at 12hz and spectral multiplexingsuited to UHPLC applications
Rapid polarity switching in both MS and MS/MSmodes
In-source CID, and HCD.
Extended mass range to 6,000m/z
Great dynamic range (>4 orders of magnitude),along with femtogram-level sensitivity
High spectral quality and a wide dynamic rangemake it possible to identify and quantify even lowabundant proteins.
Quan/qual capability make it easier to transitionfrom proteomic discovery experiments to targetedprotein quantification
Thermo Q Exactive Plus
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Thermo Q Exactive Plus
http://planetorbitrap.com
Quad
S-lens
C-trap
HCD
cell
Orbitrap
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Bottom-up proteomics
1D and 2D discovery proteomics analysis
Intact peptide and protein analysis
Analysis of PTMs
Targeted proteomics studies
Quantitation-SILAC, label-free approaches
Crosslinking studies
Untargeted metabolomics
Applications
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New, fast autotune improves sensitivity up to 5x for small molecule applications
Autotune technology also helps preserve fragile compounds for improved detection
New Agilent high voltage pulser and power supplies increase mass resolution performance and enhanced mass accuracy to 0.8 ppm
New ion optics elements provides greater robustness
New ion beam shaping optics provide more consistent results and longer usage time between cleanings
CID fragmentation
Applications: untargeted metabolomics
Agilent 6545 Q-TOF LC-MS
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Agilent 6545 Q-TOF LC-MS
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Bruker 15T FT ICR-MS
Ultimate mass resolving power in excess of RP =
10,000,000
Apollo II Ion Funnel Electrospray Source enabling high
ion transmission efficiency over a broad mass range
Optimized ion optics for sensitivity and mass range
coverage
Proprietary ParaCell ICR cell with patented SIDEKICK™
ion accumulation system for high detection efficiency.
Wide range of fragmentation techniques including CID
and ECD
Dual ionization source: MALDI source with Smartbeam II
laser and ESI source
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Bruker 15T FT ICR-MS
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High End Proteomics Studies (Top-down and Bottom-up workflows)
PTM Investigation
Accurate de novo sequencing
Molecular Imaging of Tissue—Distribution of Drugs, Metabolites, and Biomarkers
Petroleum Product Analysis
Complex Environmental Sample Analysis
Metabolomics Research
Bruker 15T FT ICR-MS applications
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Thermo Quantiva
Thermo Scientific™ active ion
management (AIM™) technology —
electrodynamic ion funnel, ion beam guide
with neutral blocker, Thermo Scientific™
HyperQuad™ quadrupole mass filter, and
active collision cell — enables attogram-
level sensitivity
Ultrafast selected-reaction monitoring
(SRM) of 500 SRM/s, with up to 30,000
definable SRMs, enables quantification of
more compounds in less time
Intuitive drag-and-drop method editor
software with application templates
simplifies method development and
operation
Applications: Targeted metabolomics
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Thermo TSQ Quantiva
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Bruker timsTOF Pro
Ion mobility time-of-flight mass spectrometer
Inclusion of IM permits differentiation between modified
peptides of same mass-different PTM sites in the same
peptide
Increased sensitivity due to ion trapping with virtually
100% duty cycles: Allows in-depth analysis from very
small sample sizes, with 50-250 ng injections routine
High dynamic range: Allows low abundance proteins to
be identified in presence of many higher abundance
proteins
High MS/MS fragmentation rate: Enables sequencing of
many peptides, and hence greater number of protein
identificationswithin shorter analysis time
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Bruker timsTOF Pro
The gas flow "behind" the ions and the electric potential allow for accumulation and staging of ions
separated by their collisional cross sections and charges.
Selected precursors are ejected from the device by scanning (lowering) the retarding potential in the
second stage
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Precursor m/z 825.9613
RT 56.08 min
Precursor m/z 825.9604
RT 55.95 min
Without ion mobility separation these overlapping
precursors would give chimeric MSMS spectra
Bruker timsTOF Pro: Co-eluting peptides
Slide courtesy of Gary Kruppa, Bruker Daltonics
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Precursor m/z 825.9613
Precursor m/z
825.9604
Without ion mobility separation these
overlapping precursors would give chimeric
MSMS spectra
• Those two peptides are co-eluted
• They are 0,0009Da apart
• Distinguishing them, based on resolution only, would have required
a resolving power > 900 000 !
• … and that alone would not have allowed to isolate them
separately
PASEF DOES IT…
@ > 100 Hz MS/MS
While preserving sensitivity !
Bruker timsTOF Pro: Co-eluting peptides
Slide courtesy of Gary Kruppa, Bruker Daltonics
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Bruker timsTOF Pro: Iosbaric peptidesTIMS separation of co-eluted isobaric HEK Phosphopeptides
• Similar seqences
• Several Phosphoryation sites
on the sequence
• Probable isoforms
Slide courtesy of Gary Kruppa, Bruker Daltonics
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New services
Proteomics Quantitation-label free, TMT, SILAC
2D LC/MSMS for Protein Identification and PTM Study on both Orbitrap Fusion and QE
MADLI Tissue Imaging
Top-down Proteomics
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Slide courtesy of Nilini S. Ranbaduge
High pH fractionation with Low pH separation
Dionex 3000 2D System
$60/fraction on the orbitrap XL
$75/fraction on the orbitrap Fusion or
Q Exactive
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Fractionation (F)
pH 10.0
Gradient separation19
RT: 0.00 - 90.00
0 10 20 30 40 50 60 70 80 90
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
45.29
45.96
43.94
38.87
54.28
61.83
30.0765.4629.06
25.19
22.86 68.9071.054.27 21.37 87.65
NL: 2.71E8
Base Peak F: FTMS + p NSI Full ms [400.00-1600.00] MS NR_20141223_HCC15_pooled_test
RT: 0.00 - 89.99
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
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100
Rel
ativ
e A
bund
ance
NL: 2.89E8
Base Peak F: FTMS + p NSI Full ms [400.00-1600.00] MS NR_20150321_A549T_LKB-FT_test
RT: 0.00 - 79.99
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
NL: 2.31E8
Base Peak F: FTMS + p NSI Full ms [400.00-1600.00] MS NR_20150201_A549_LKB-P_15F_80min_F2
RT: 0.00 - 80.00
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abun
danc
e
NL: 2.74E7
Base Peak F: FTMS + p NSI Full ms [400.00-1600.00] MS NR_20150201_A549_LKB-P_15F_80min_F15
F1
F2
F15
Slide courtesy of Nilini S. Ranbaduge
High pH fractionation with Low pH separation
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Top Down vs Bottom up
Pflugers Archiv-European
Journal of
Physiology, 2014, 466, 1199-
1209.
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m/z750730710690670650630610590570550
c17+84
c26+70
c26+112
c12+42
c22+112
c12+42
c13+42
c25+112
c23+112
c40+70
c40+112
C24+70
c24+112
c7+42
c15+42
c37+70
c55+112
c37+140
c37+112
c38+112
c26+70
c26+112
c52+70
c52+112
c54+70
c54+112
c18+84
z35+14
z35+56
z12
c59+70
c59+112
z20
C60+112
m/z750730710690670650630610590570550
c17+84
c26+70
c26+112
c12+42
c22+112
c12+42
c13+42
c25+112
c23+112
c40+70
c40+112
C24+70
c24+112
c7+42
c15+42
c37+70
c55+112
c37+140
c37+112
c38+112
c26+70
c26+112
c52+70
c52+112
c54+70
c54+112
c18+84
z35+14
z35+56
z12
c59+70
c59+112
z20
C60+112
1000900800700600 780770760750
m/z
+1Ac + 2Me
+1 Ac+ 4Ac
+ 5Ac
+ 2Ac + 2Me
Parents ions (750 - 770, 15+) 758.0864 15+
754.7814 15+
+ 4Ac
- 15+
15+
1000900800700600 780770760750
m/z
+1Ac + 2Me
+1 Ac+ 4Ac
+ 5Ac
+ 2Ac + 2Me
Parents ions (750 - 770, 15+) 758.0864 15+
754.7814 15+
+ 4Ac
- 15+
15+
ECD MS/MS
m/z1000990980970960950940930920910900
z32
z48
z64
z49+14
z9
z65
z49z66
z25+16
z50
z34
z67
z76
z42
z42+14
z59
z51
z68
z43
z78z61 z79
z62
z27z75
m/z1000990980970960950940930920910900
z32
z48
z64
z49+14
z9
z65
z49z66
z25+16
z50
z34
z67
z76
z42
z42+14
z59
z51
z68
z43
z78z61 z79
z62
z27z75
Histone H4
Slide courtesy of Liwen Zhang
Protein Identification by Top Down MS/MS
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N-AGRGK5GGK8GLGK12GGAK16RHRK20VLRDNIQGIT H4
Ac Ac Ac Ac Ac
Me Me
Ac Ac Ac
Known Modification
Modification Detected
Protein Identification by Top Down MS/MS
$500 on Fusion or ICR
Slide courtesy of Liwen Zhang
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Large molecules >10,000 Da
$750/sample TOF-TOF
Small molecules <10,000 Da
$750/Sample on 15T ICR
Imaging Mass Spectrometry
• High Mass Accuracy (FT ICR)
assists in chemical formula
determination
• Changing Matrix can help with
interference
• Derivatization for unstable
molecules
Anal. Chem., 2011, 83 (24), 9694–9701
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RT: 40.31 - 210.44
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210
Time (min)
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Re
lative
Ab
un
da
nce
0
10
20
30
40
50
60
70
80172.3651.09 74.43
147.2873.4659.81
83.0067.5146.94
84.43125.6190.98
98.24
102.59 154.41135.68
107.81113.21 129.55 156.09140.88 161.85121.24 203.90186.96 193.16175.36
96.30
74.03
72.89 97.42 164.8778.17 82.2072.6559.3690.1855.88 185.19123.9166.13 128.35
101.89153.68113.47 188.09 199.8052.73
50.86 130.21 163.13141.80 195.09178.09 208.48120.3046.39
146.51171.8573.23
171.2978.49
97.0378.6667.29
56.1859.48 82.84 85.96 128.6398.06 185.93113.7690.37
168.18124.41 189.02 200.9953.11 153.97105.4151.41124.14 135.09 155.6747.08
210.16184.39 200.06120.75
NL:9.28E7
Base Peak MS 145-300-1
NL:8.28E7
Base Peak MS 145-3000-2
NL:1.15E8
Base Peak MS 145-3000-3
Slide courtesy of Liwen Zhang
Laser Capture Microdissection Proteomics
Laser capture
microdissection
available through the
laser capture
molecular core
https://lcm.osu.edu/
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Additional facility MS instrumentation
Thermo Fusion
Thermo Orbitrap XL
Thermo LTQ
Bruker ultrafleXtreme MALDI-TOF TOF
Bruker amaZon ion trap with ETD
Bruker maXis UHR QTOF
Thermo DSQ II GC-MS
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http://www.ccic.ohio-state.edu/MSP