The ability to fully characterize proteins in their intact forms allows thorough biological investigation of the functional importance of changes such as post-translational modifications, protein isoforms/sequence variations, and protease cleavages.

1. Identification and characterization of intact proteins in complex mixtures using online fragmentation on the new Orbitrap Elite
Shannon Eliuk1, John F. Kellie2, David Horn1, Neil Kelleher2, Vlad Zabrouskov1
Thermo Fisher Scientific, San Jose, CA 95134, USA1; Northwestern University, Evanston, IL 60208, USA2
M th dO i L li ti f M difi ti Id tifi ti f l t iMethodsOverview
Purpose: To evaluate the performance of the new Orbitrap Elite hybrid
mass spectrometer for the in-depth top down analysis of complex
intact protein mixtures
Figure 2: Typical Workflow for Top Down Proteomics
yeast cell lysate
GELF EE i t t t i f ti ti
[ ]
743.6234
Localization of Modifications
Identification of 60S Acidic Ribosomal Protein P2-β
and localization of phosphorylation site
Identification of larger proteins
Multiple fragmentations improved fragment ion coverage for larger proteins
Phosphoglycerate mutase 1 Triosephosphate isomerase
]
889.7448
R=74302
z=30
834.2616
R=73123
z=32
953.2260
R=72067
z=28809 0115intact protein mixtures.
Methods: The protein identification capabilities of the Orbitrap Elite by
LC-MS/MS were investigated with the use of a fractionated yeast
lysate. Identifications were based on accurate, high resolution mass
t f b th th d d t i I d t
(200 – 500 μg)
reduced/
denatured
–
GELFrEE intact protein fractionation
10kDa
25kDa
1113.9489
R=71900
z=101012.8633
R=74600
z=11
1012.4087
R=74204
z=11
R=79036
z=38
786.0863
R=74685
z=35
724.0806
R=78538
z=38
833.5759
R=75922
z=33
859.6249
R=74062
z=32
916.9332
R=68687
z=30
705.5661
R=79077
+phosphorylation
+phosphorylation
11,123.3 Da
MS/MS of +11 charge state
27,460.6 Da
MS/MS of +38 charge state
p p
26,647.9 Da
MS/MS of +35 charge state
z 28
988.4935
R=71250
z=27785.2181
R=74884
z=34
809.0115
R=75663
z=33
762.7830
R=78458
z=35
30 kDa proteins are baseline
resolved with a single scan bymeasurements of both the precursor and product ions. Improved top
down characterization of proteins was evaluated using multiple
fragmentation techniques including CID, HCD, and ETD. Data were
analyzed using ProsightPC 2.0.
(200 500 μg)
+
10kDa
Sample clean up
(SDS removal)
O li LC MS/MS
1012.8633
R=74600
z=11
928.5427
1014.2279
R=73604
z=?
1013.2281
R=74304
z=11
1014.5928
R=71704
z=?
R 79077
z=39
948.4468
R=70720
z=29
982.2840
R=71910
z=28
687.9270
R=78750
z=40
1057.7686
R=65345
?
671.2219
R 82924 1104 8111
+oxidation
z 35
741.6783
R=77626
z=?
721.5791
R=80160
z=?
702 7481
resolved with a single scan by
LC-MS/MS. The online
fragmentation enables accurate
identification of the intact proteins.
Results: The Orbitrap Elite hybrid mass spectrometer was very
successful at identifying proteins in a complex mixture using online LC
separation. The improved resolution and scan speed allowed the
accurate identification of proteins with sensitive detection of fragment
i i h i d f i Th bi i f l i l
High-throughput
data analysis
Online nanoLC-MS/MS
on an Orbitrap Elite with ETD
&
R=77100
z=12
857.1167
R=79200
z=13
962.1617
R=75800
z=18
911.5227
R=76604
z=?
879.3952
R=81304
1154.3920
R 67500
1012 1014 1016
m/z
1016.3146
R=66800
z=11
1015.5927
R=65004
z=11
1011.3165
R=73004
z=?
700 800 900 1000 1100 1200
m/z
z=?R=82924
z=2
1195.7383
R=63902
z=24
655.2403
R=82195
z=2
1104.8111
R=65263
z=? 743.5
m/z
762.5 763.0
m/z
700 750 800 850 900 950 1000
m/z
702.7481
R=77021
z=37
Sample: Yeast lysate (Sigma-Aldrich).
Lysate Desalting: The necessary desalting, required before GELFREE
ions in a short period of time. The combination of multiple
fragmentation types improved the sequence coverage and allowed the
localization of post-translational modifications such as phosphorylation.
850 900 950 1000 1050 1100 1150
m/z
z ?R 81304
z=?
R=67500
z=15
m/z
ETD CID HCD
ETD – 21 fragments CID – 17 fragments HCD – 23 fragments
y g y g q
fractionation, was performed using the Thermo Fisher Scientific Zeba Spin
Desalting columns with a 7 kDa MWCO (product # 89890).
GELFREETM Fractionation of Intact Protein Lysate: Fractionation of proteins in
the mass range of 3.5-100 kDa was performed with a GELFREE 8100 10% Tris-
A t t C t id Kit th GELFREE 8100 F ti ti S t (P t i
Introduction
The ability to fully characterize proteins in their intact forms allows thorough
biological investigation of the functional importance of changes such as post- 400 600 800 1000 1200 1400 550 600 650 700 750 800 850 900 950 1000 200 400 600 800 1000 1200 1400
ETD CID HCD
Acetate Cartridge Kit on the GELFREE 8100 Fractionation System (Protein
Discovery) according to the manufacturer’s protocol.
Detergent Cleanup: Using the Thermo Scientific Pierce Detergent Removal
Resin (product # 87777), we efficiently removed the detergents introduced for the
GELFREE separation
biological investigation of the functional importance of changes such as post
translational modifications, protein isoforms/sequence variations, and protease
cleavages. Characterization of proteins at the intact level by nanoLC-MS/MS has
long been of interest for mass spectrometrists as this technique can provide
essential information, often in a non-targeted fashion, regarding modification
chemistries localized to specific residues Many of these findings are otherwise
400 600 800 1000 1200 1400 1600 1800 2000
m/z
600 700 800 900 1000 1100 1200 1300 1400
m/z
200 400 600 800 1000 1200 1400 1600 1800 2000
m/z
400 600 800 1000 1200 1400
m/z
550 600 650 700 750 800 850 900 950 1000
m/z
200 400 600 800 1000 1200 1400
m/z
ETD – 20 fragments CID – 17 fragments HCD – 30 fragments
GELFREE separation.
Online LC: Fractions 1 and 4 were analyzed by further LC-MS/MS as described
here. Intact proteins were separated online using the split-free EASY-nLC
(Thermo Scientific) with solvent compositions as follows: solvent A: 0.1% formic
acid in water, solvent B: 0.1% formic acid in acetonitrile. The gradient consisted
chemistries localized to specific residues. Many of these findings are otherwise
not easily discernable using standard proteomics practices involving protein
digestion, this includes the ability to discern protein isoforms, localize
combinations of post-translational modifications, and determine in vivo protease
cleavage sites. , g
of 5-30% solvent B over 10 min, 30-65% solvent B over 45 min, and 65-90%
solvent B over 5 min. The proteins were separated using a 5 μm, 1000 Å polymer
(polystyrene divinylbenzene) reversed phase (PLRP-S) precolumn (3 cm x 150
μm ID) and analytical column (10 cm x 75 μm ID) purchased from New Objective.
Here we introduce a newly developed Thermo Scientific Orbitrap Elite hybrid
mass spectrometer which combines unprecedented scans speeds at extremely
high resolution (>240,000) with high sensitivity and accurate mass characteristic
of our hybrid Orbitrap platforms (Figure 1). The high-field Orbitrap and advanced
signal processing has improved the analysis of level intact proteins in relatively
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
400 600 800 1000 1200 1400
m/z
500 600 700 800 900 1000 1100 1200 1300
m/z
Site of phosphorylation
(out of 9 modifiable residues)
Site of incomplete oxidation
MS Analyses: On the new Thermo Scientific Orbitrap Elite hybrid mass
spectrometer, the resolution was set to 100K for full scan with 8 μscans and 1E6
AGC target. The resolution was set to 60K for MS2 with 10 μscans and 2E6 AGC
target. The maximum IT was 2s for both full scan and MS2 scan. ETD, HCD, and
CID fragmentation was performed on the most intense ion with the m/z as
signal processing has improved the analysis of level intact proteins in relatively
complex mixtures on an LC timescale. In this study we evaluated a complete top
down proteomics workflow from sample preparation to data analysis providing a
robust and sensitive method for identification and characterization of proteins in a
mixture using a variety of fragmentation techniques including CID, HCD, and
ETD
Summary and Conclusions
 The Orbitrap Elite is a top of the line mass spectrometer for the identification of
intact proteins on an LC timescale
 High resolution MS allowed baseline resolution of isotopes for intact713.9760
Fragmentation of small proteins
Full sequence characterization from both N and C termini
ETD – 20 msec reaction time ETD – 5 msec reaction time
masses option selected to only perform MS/MS on the most intense charge state
per protein. The protein identifications were performed with ProSightPC 2.0
software (Thermo Scientific).
ETD.
g p
proteins up to 30 kDa with low ppm mass accuracy
 50 kDa proteins can be baseline resolved with resolution of 240,000
 Multiple modes of fragmentation (CID, ETD, and HCD) improved fragment
ion coverage of intact proteins and allowed localization of sites of
difi tiFIGURE 1 Schematic of the new Orbitrap Elite hybrid mass spectrometer N hi h fi ld O bit ith
R=88900
z=12
778.9737
R=84700
z=11
659.1323
R=90300
z=13
856 6701
Ubiquitin
8,551.6 Da
MS/MS of +13 charge state
modifications
 Faster scan speeds at high resolution settings (60K resolution at ~4 Hz)
allowed more microscans to be summed on the LC-timescale to improve
both MS and MS/MS analyses
 Improves signal to noise and ion statistics of intact proteins and
FIGURE 1. Schematic of the new Orbitrap Elite hybrid mass spectrometer
S-lens
Square Quadrupole
with Beam Blocker
Octopole
Quadrupole
Mass Filter
High
Pressure
Cell
Low
Pressure
Cell C-Trap
HCD
Collision Cell
Transfer
Multipole
Reagent Ion
Source
• New high-field Orbitrap with
advanced signal processing
providing more scans at a
given resolution (>4 Hz at
60,000 resolution vs 1 Hz on
856.6701
R=82700
z=10
794.3848
R=84301
z=1 1136.5524
R=71201
z=1
930.9467
R=77000
z=17
1031.6865
R=70700
z=15
911 4166
659.0 659.5
m/z 400 600 800 1000 1200 1400 1600
m/z
200 400 600 800 1000 1200
m/z
Combination of multiple
fragmentation techniques,
including multiple reaction times
for ETD, provided extensive
 Improves signal to noise and ion statistics of intact proteins and
fragment ions
 Improves fragment ion coverage in less time
 Ion trap accumulation prior to MS/MS allowed identification of low intensity
proteins with high quality spectraNew High-Field Orbitrap
LTQ Orbitrap Velos) and a
higher maximum resolving
power of 240,000.
• Robust and sensitive 600 700 800 900 1000 1100 1200
m/z
911.4166
R=79704
z=?
824.9016
R=79104
z=?
631.2975
R=87804
z=?
758.5731
R=83104
z=?
CID HCDsequence coverage with single
scan LC-MS/MS
p g q y p
References
Lee, Kellie, et al. A robust two-dimensional separation for top-down tandem mass
spectrometry of the low-mass proteome J Am Soc Mass Spectrom 2009
g p
Mass Analyzer
**Increased resolution and scan
rate for higher quality top down
data, compatible with LC
timescale**
generation II ion optics with
beam blocking.
• New pre-amplifier for
improved S/N. 450 500 550 600 650 700 750 800 850 900 950 200 300 400 500 600 700 800 900 1000
spectrometry of the low mass proteome. J. Am. Soc. Mass. Spectrom. 2009
Dec;20(12):2183-91
m/z m/z