Application
Note: 30217
1000 Proteins per Hour [pph] Identified from
an E. coli Digest
Maximizing Protein Identification on the LTQ Orbitrap Velos
Kai Scheffler, Thermo Fisher Scientific, Dreieich, Germany
Eugen Damoc, Thomas Möhring, Thermo Fisher Scientific, Bremen, Germany
Introduction
Key Words
•LTQ Orbitrap
Velos
•CID, ETD, HCD
•DDDT, MS/MS
• E. coli
•Proteome
Discoverer
•Protein
Identification
Enormous improvements in mass spectrometry technology
over the last few years have allowed for ever deepening
analysis of complex proteomes. However, many low abundance proteins remain undetected in current large-scale
proteomic analyses due to the limits on the rate at which
MS/MS spectra can be acquired. To solve this undersampling problem, we developed the Thermo Scientific
LTQ Orbitrap Velos, a new hybrid linear ion trap-Orbitrap
mass spectrometer that provides faster MS/MS scanning
in the ion trap (up to 10 peptide sequences per second),
thereby increasing the depth of analysis of complex protein
mixtures.
This application note demonstrates and discusses the
ability of the newly developed hybrid instrument to identify more proteins, with increased sequence coverage and
confidence. Different strategies regarding chromatographic
run times and utilizing the instruments various dissociation
techniques are compared.
Instrument Innovation
The instrument evaluated in this application note is the
Thermo Scientific LTQ Orbitrap Velos mass spectrometer
equipped with Electron Transfer Dissociation (ETD). It is
a hybrid instrument which includes a novel dual-pressure
linear ion trap combined with an Orbitrap™ detector as
shown in Figure 1.
Significant technological improvements have been implemented in both parts of the hybrid instrument. Changes
implemented in the front-end of the hybrid instrument,
the Thermo Scientific LTQ Velos ion trap, include (a) a
progressive stacked-ring ion guide (S-Lens) with 5-10x
increased ion transmission;1 (b) a dual-pressure differentially pumped linear ion trap with a higher-pressure cell for
improved ion trapping, isolation, and CID efficiencies, and
a lower pressure cell for improved resolution and/or scan
speed and (c) predictive automatic gain control (AGC) for
increased scan speed.2 Electrospray Ion Source
S-Lens
Square Quadrupole
Octopole
Improvements in the rear portion of the instrument,
the Orbitrap mass analyzer section, are: (d) an HCDcollision cell with axial field combined with the C-trap and
(e) improved vacuum in the Orbitrap detector chamber for
improved analysis of intact proteins.3
Experimental
Sample Preparation
The lyophilized soluble fraction of an E. coli whole cell
lysate was solubilized in 50 mM ammonium bicarbonate buffer followed by reduction of disulfide bonds with
dithiothreitol (DTT) at 56 °C for 1 hr, and alkylation of
cysteines with iodoacetamide for 30 min at room temperature in the dark. The sample was then enzymatically
digested for 14 hours with a Lys/Arg specific protease. The
digest was split in aliquots and stored at -20 °C until use.
Samples were diluted in 0.1% formic acid prior to analysis
by reversed-phase nanoHPLC-MS/MS.
LC-MS Analysis
The proteolytic digest of E. coli was separated by online reversed-phase chromatography for each run using a
Thermo Scientific Surveyor MS Pump Plus and Micro AS
autosampler with a reversed-phase peptide trap (100 μm
inner diameter, 2 cm length) and a reversed-phase analytical column (75 μm inner diameter, 10 cm length, 3 μm
particle size, both NanoSeparations, NL), at a flow rate
of 250 nl/min. The chromatography system was coupled
on-line with an LTQ Orbitrap Velos mass spectrometer
equipped with an ETD source.
High Pressure Cell Low Pressure Cell Quadrupole Mass Filter
C-Trap
HCD Collision Cell
Transfer Multipole
Reagent Ion Source
e-
Orbitrap
Mass Analyzer
Reagent 1
Heated Inlet
Figure 1: Schematic of the Thermo Scientific LTQ Orbitrap Velos mass spectrometer with ETD.
Reagent 2
Heated Inlet
Instrument Configuration
Pump
Thermo Scientific Surveyor MS Pump Plus
Autosampler
Thermo Scientific Micro AS
Mass spectrometer
Thermo Scientific LTQ Orbitrap Velos with ETD
Ionization source
Nanospray I
Trapping column
20 mm in length, 100 µm ID
Analytical column
100 mm x 75 µm ID
3 µm particle size, 300 Å pore size
Flow rate
250 nl/min at 50% B
Gradient
2-30% acetonitrile in 0.1% formic acid
(either 60 or 90 minutes)
Table 1: Instrument configuration.
Mass Spectrometer Settings
Sourcenano-ESI
Capillary temperature
250 °C
S-Lens RF level
50%
Source voltage [kV]
1.5
Max. injection times
Full MS 500ms
MSn
50ms (CID, ETD)
MSn
200ms (HCD)
Full MS mass range
380 - 2000 [m/z]
Details for the chromatographic and mass spectrometric settings are listed in Table 1 and 2. Dilutions of the
injected sample amounts were performed to evaluate the
effects of decreasing sample quantity in regards to sensitivity of the instrument as judged upon the total number of
proteins identified as well as their sequence coverage.
Data-dependent tandem MS acquisition methods were
used for all experiments. Different fragmentation techniques, namely CID, ETD and HCD and combinations
thereof by using the data dependent decision tree (DDDT)
acquisition method were compared.4 All runs were performed at least in duplicates. In all
experiments the Full MS scans were acquired over a mass
range of 380-2000 m/z with detection in the Orbitrap mass
analyzer at a resolution setting of 30,000. MS/MS spectra
were acquired in the ion trap except for fragment ions produced using higher-energy collision-induced dissociation
(HCD) taking place in the collision cell positioned in axial
position next to the C-trap.
Fragment ion spectra produced via HCD were acquired in the Orbitrap mass analyzer with a resolution setting of 7,500. For data-dependent acquisition, the method
was set to analyze the top twenty most intense ions when
fragmentation was performed using CID (Table 3).
Resolution settings
Full MS 30.000
MSn (HCD) 7.500
Lock mass (all runs)
445.120024
Table 2: Mass spectrometer parameter settings.
Fragmentation Type
CID
ETD
HCD
DDDT
Isolation width [u]2222
Collision energy30n.a.4030 (CID)
Activation time [ms]101000.110 (CID) / 100 (ETD)
Supplemental activationn.a.yesn.a.yes (ETD only)
Pred. AGC enabledyesyesyesyes
No. of microscans for Full MS2212
Target value Full MS (FT)1.00E+061.00E+061.00E+061.00E+06
Detector for MSn spectraIon TrapIon TrapOrbitrap
Ion Trap
Target value MSn5.00E+035.00E+035.00E+045.00E+03
Top n MS/MS, with n =20101010
Charge state dep. ETD time enabledn.a.yesn.a.yes
Dynamic exclusion enabledyesyesyesyes
General Settings Dynamic exclusion settings
repeat count 1
exclusion list size 500
exclusion duration 70 s
early expiration disabled
exclusion mass width low/high 10 ppm
Charge state screening on: +1 and unassigned charge states rejected
Monoisotopic precursor selection enabled
Table 3: Method parameter settings.
2
The top ten most intense ions per scan cycle were
fragmented in case of ETD spectra and also when the
DDDT method was applied. Parameters for the CID/ETD
data dependent decision tree were set to perform ETD
when the precursor charge state was z=3 and m/z of the
precursor < 650 u, with charge state z=4 and m/z < 900 u
and charge state z=5 with m/z < 900 u. In all other cases
peptide fragmentation was triggered with CID.
For data-dependent acquisition of HCD spectra, the
top ten most intense ions were selected for fragmentation
in each scan cycle and Full MS as well as fragment ion
spectra were detected in the Orbitrap mass analyzer. Exclusion conditions were optimized according to observed
chromatographic peak width.
Due to the greater ion transmission of the ion source,
a lower injection time was selected for all MS/MS spectra
detected in the ion trap: 50 ms compared to 100-200 ms
on previous instruments. The activation time for resonance
CID fragmentation was 10 ms compared to 30 ms used
on the Thermo Scientific LTQ XL linear ion trap, taking
advantage of the increased fragmentation efficiency in the
higher-pressure cell of the ion trap. For HCD experiments
a lower target value of 5e4 ions was used compared to 2e5
on previous instruments, taking the advantage of better
transmission of fragment ions from the axial field collision
cell.
Database Search
The raw data files were searched using Thermo Scientific
Proteome Discoverer software v. 1.2 with Mascot™ v. 2.2.1
search engine (Matrix Sciences Ltd., London, UK). The
search parameters applied in the database searches are
listed in Table 4. The reverse database search option was
enabled in Mascot, and all data were filtered to satisfy a
false discovery rate (FDR) of 1% or better. All datasets
were processed using the workflow feature in Proteome
Discoverer™ software. The workflow used to process
datasets containing ETD spectra included the node called
non-fragment ion filter which aims at the removal of intact
precursors and its charged reduced species since they are
very often the most abundant peaks in the fragment ion
spectra und thus compromise the score.
Protein Annotation
All identified proteins from the duplicate runs from CID,
ETD, HCD and DDDT methods were exported from the
Proteome Discoverer software as ProtXML format and
submitted to the Thermo Scientific ProteinCenter data
interpretation software to retrieve relevant biological information from publicly available protein databases including
gene ontology references for function, biological process
and subcellular location.
Software Platform
Search engine
Proteome Discoverer
Mascot v 2.2.1
DatabaseSwissProt v 56.0
TaxonomyEscherichia coli
Maximum number of
missed cleavages1
Precursor mass tolerance
5 ppm
Fragment mass tolerance
0.8 Da (CID, ETD), 20 mmu (HCD)
Protein massunrestricted
Reverse database search
enabled
Target false discovery rate
0.01 strict/0.05 relaxed
Instrument typeDefault
Modifications
static: carbamidomethyl cysteines (C)
Scoring
Peptide cut off score: 10
MUDPIT scoring
Protein relevance threshold: 20
Protein relevance factor: 1
DTA Generation conditions
Total intensity threshold: 100
Minimum peak count: 8
S/N threshold: 2
Precursor mass range: 760 - 10.000 Da
Non-fragment filter
(for ETD spectra only)
Mass window offset
for precursor peak: 4 Da
Mass window offset
for charge reduced precursors: 2 Da
Mass window offset for neutral
losses from charge reduced
precursors: 2 Da
Remove only known masses: false
Maximum neutral loss mass: 120 Da
Result Filters
Confidencehigh
Min. number of peptides per protein 0/2
FDRfixed to 1%
Table 4: Data analysis parameter settings.
Results and Discussion
The LTQ Orbitrap Velos mass spectrometer identifies more
than 1,000 proteins per hour [pph]
Figure 2 illustrates the base peak chromatograms from
analyses of 1,000 ng, 100 ng and 20 ng of a proteolytic
digest of E. coli whole cell lysate, separated over 60 min
via reversed-phase chromatography on the LTQ Orbitrap
Velos mass spectrometer with a split flow from a Surveyor
MS pump. The peak intensities in the chromatograms
correlate very well with the sample amount analyzed and
also demonstrate the moderate complexity of the sample
compared to more complex lysates from other organisms
such as C. elegans2 or human HeLa cells.3
3
514.30
Relative Abundance
100
442.95
80
652.40
421.76
562.28
449.74
607.82
701.37
519.93
15
20
25
30
35
849.75
806.45
45
50
Time (min)
55
718.38
544.29
60
65
70
75
601.97
442.95
688.82
100 ng
586.33
423.78
609.79
594.26
40
652.40
562.28
463.91 449.74
20
15
607.82 538.79
20
25
30
35
40
637.61
688.82 609.79
20
806.45
60
20 ng
748.43
423.78
701.37
25
30
35
75
652.40
607.82
609.69
543.81
735.90
803.45
624.37
20
70
NL: 8.80E6
Base Peak
F:ms MS
445.12
15
65
594.32
443.20
562.28
55
483.02
594.26
60
449.74
823.91
706.73
586.33
601.97
80
859.11
983.48
45
50
Time (min)
514.30
100
40
NL: 3.26E7
Base Peak
F:ms MS
594.32
514.30
60
0
609.35
748.43
595.04
80
0
40
982.02
983.48
613.32
20
100
Relative Abundance
1 µg
859.11
40
NL: 2.89E8
Base Peak
F:ms MS
706.73
60
0
Relative Abundance
601.97
688.82
706.73
594.32
586.33 423.78
40
45
50
Time (min)
55
723.40
60
445.12
65
445.12
445.12
70
75
Figure 2: Base peak chromatograms of 1 µg (top trace), 100 ng (middle trace) and 20 ng (bottom trace) of E. coli proteolytic digest loaded on a 75 µm C18 reversed phase column and separated via a 60 min gradient (2-30% ACN).
For a typical and reproducible 60-minute separation
the LTQ Orbitrap Velos mass spectrometer is able to identify over 1,000 proteins from 1 µg of a proteolytic digest
of E. coli whole cell lysate using a ddTOP20 CID method
with acquisition of the full scan in the Orbitrap detector
and the fragment ion spectra in the ion trap. Reducing the
sample amount by a factor of 10 to a final 100 ng applied
on column, the number of identified proteins is only reduced by about 10%. Reducing the sample amount again
by a factor of 5 to 20 ng, the number of identified proteins
is again reduced by about 10%, resulting in 844 proteins
on average as depicted in Figure 3a.
(a)
1200
1046
1054
1000
800
Proteins
Proteins with ≥ 2 Peptides
942
789
859
829
809
683
600
947
689
577
567
400
200
0
4
1000
100
Sample Amount Loaded [ng]
20
The total number of proteins identified in the duplicate
runs for the three sample concentrations are very close and
the overlap of the identified proteins was 90% across all
concentrations indicating high reproducibility. Identification rate on the peptide levels demonstrates a success rate
of identified peptides relative to the total number of CID
spectra acquired of ~53% for all concentrations. The percentage of unique peptides identified versus all identified
peptides varies between 70% (1 µg load) and 90% (20 ng
load) as displayed in Figure 3b.
(b)
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
All Peptides
Unique Peptides
9833
9257
6424
6740
6504
5356
6432
5322
4282
4165
3736
1000
100
Sample Amount Loaded [ng]
3793
20
Figure 3: Identification of unique proteins (a) and identification of unique peptides (b) from 1 µg/100 ng/20 ng of E. coli proteolytic digest
using the 60 min ddTOP20 CID method. For a discussion on: “False discovery rates of protein identification: A strike against the two-peptide rule.” see7.
The LTQ Orbitrap Velos offers great acquisition speed
The high numbers of identified peptides and proteins using
the LTQ Orbitrap Velos system is strongly based on the
high acquisition speed that the instrument offers. Figure 4
highlights the speed at which spectra are acquired. Every
vertical line in the diagrams represents a scan resp. a spectrum in the chromatogram. The green lines represent the
Full MS scans, whereas the orange lines represent the
MS/MS spectra. In the ddTOP20 CID method (Figure 4,
top) the time elapsed for the acquisition of a Full MS spectrum is 1.13 sec due to the acquisition with 2 µscans and
a resolution setting of 30k. The time elapsed to acquire
20 CID MS/MS spectra is 2.36 sec resulting in 3.49 sec for
a full scan cycle comprising of 1 Full MS spectrum (Orbitrap detector) and 20 MS/MS spectra (ion trap detector).
RT: 29.488 - 29.600
0.10
0.08
0.06
0.04
0.02
0.00
Relative Abundance
29.507
0.10
0.08
0.06
0.04
0.02
0.00
1.13 sec
29.50
29.51
29.52
29.53
29.54
29.55
Time (min)
45.597
1.0
0.5
29.56
29.585
45.591
Full MS/MS trace
TOP20 CID
29.57
29.59
29.60
Full MS trace
30k, 1 µscan
2.29 sec
45.657
45.607
45.613
45.620
45.628
45.635
45.609
45.616
45.624
45.632
45.640
1.0
Full MS/MS trace
TOP10 HCD, 7.5k
0.5
0.0
45.57
29.58
45.645
HCD
0.64 sec
Relative Abundance
Full MS trace
30k, 2 µscans
2.36 sec
29.526
29.563
29.561
29.528
29.559
29.529
29.556
29.531
29.554
29.533
29.552
29.535
29.550
29.537
29.549
29.539
29.547
29.541 29.545
29.542
RT: 45.570 - 45.680
1.5
0.0
1.5
29.565
CID
29.504
29.49
In higher energy collision induced dissociation (HCD),
a dissociation technique that has proven to be especially
useful for de novo sequencing as well as for quantitation using chemical labeling, all scans are detected in the
Orbitrap mass analyzer at high mass accuracy and a high
resolution setting of choice between 7,500 and 100,000.5,6
The cycle time of a ddTOP10 HCD (Figure 4, bottom) is
2.93 sec, comprising of 0.64 sec for the Full MS acquired
with 1 µscan and 2.29 sec for 10 MS/MS HCD spectra
recorded at a resolution setting of 7,500. These two
examples show the higher speed at which CID and HCD
spectra can be acquired compared to the LTQ Orbitrap XL
mass spectrometer, which is due to the implemented hardware improvements.3
45.58
45.59
45.60
45.61
45.62
45.63
Time (min)
45.64
45.65
45.66
45.67
45.68
Figure 4: Demonstration of scan speed using a ddTOP20 CID method (top) compared to a ddTOP10 HCD method (bottom).
With CID 1.13 sec are spent on the acquisition of the Full MS scan followed by 2.36 sec for 20 MS/MS spectra acquired in the ion trap.
With HCD 0.64 sec are spent on the Full MS scan followed by 2.29 sec on 10 HCD MS/MS spectra acquired at a resolution of 7,500.
omparing different fragmentation techniques for maxiC
mizing protein ID
The LTQ Orbitrap system offers various different fragmentation techniques respectively methods. Whereas CID,
PQD (pulsed Q Dissociation) , ETD and HCD are fragmentation techniques, DDDT is an instrument method.
The choice which of the available fragmentation methods is most suitable for the analysis very much depends
on the complexity and the amount of sample available for
repetitive runs, the protease used for digestion, and last but
not least the aim of the analysis such as purely protein ID
or identification combined with quantitation, de novo sequencing or the analysis of post-translation modifications.
In this current study the aim was to identify the maximum
number of proteins using a standard digest with a K,Rspecific endoprotease.
Figure 5 summarizes the results regarding peptide and
protein identification resulting from four of the possible
fragmentation modes run as ddTOP20 CID, ddTOP10
DDDT, ddTOP10 ETD and ddTOP10 HCD. All methods
were run in duplicate runs with 1 µg and 100 ng sample
amount each with a separation of the peptide mixture
over the course of 90 min. The numbers presented in the
diagram are the average of the duplicate runs. All methods
applied show a very high number of unique peptides as
well as unique proteins identified with CID leading the
field with an additional ~5% of proteins identified compared to the DDDT method. Overlap of proteins identified
with all four methods is about 80% (Figure 6).
5
(b) Unique Peptides
8000
(a) Proteins
7000
1200
6000
1000
800
600
5000
1157 995
1095 957
1017 806
914
832
7601 5858
7009 5603
6344 4759
5577 4595
1 µg 100 ng
1 µg 100 ng
1 µg 100 ng
1 µg 100 ng
CID
DDDT
HCD
ETD
3000
400
2000
200
0
4000
1000
1 µg 100 ng
1 µg 100 ng
1 µg 100 ng
1 µg 100 ng
CID
DDDT
HCD
ETD
0
Figure 5: Identification of unique proteins (a) and identification of unique peptides (b) from 1µg and 100 ng E. coli digest using 90 min LC separation and
different methods: ddTOP20 CID, ddTOP10 DDDT, ddTOP10 HCD and ddTOP10 ETD
(a) Total of 8825 Unique Peptide IDs at < 1% FDR and < 5ppm
(a) Total of 1268 Protein IDs
CID
158
CID/HCD
134
HCD
520
HCD
39
CID/HCD/ETD
851
CID/HCD/ETD
4397
HCD/ETD
348
HCD/ETD
14
CID/ETD
26
CID/HCD
1378
CID
1338
CID/ETD
523
ETD
46
(b) Total of 1306 Protein IDs
(b) Total of 9069 Unique Peptide IDs at < 1% FDR and < 5ppm
CID/DDDT
169
CID/DDDT
1431
CID
123
DDDT
77
CID
1285
CID/ETD/DDDT
857
CID/ETD
21
ETD
321
CID/ETD/DDDT
4493
ETD/DDDT
17
ETD
42
CID/ETD
427
DDDT
764
ETD/DDDT
365
ETD
304
Figure 6: Venn diagrams summarizing the results from three 90 min nanoLC-MS/MS runs of 1 µg E. coli whole cell lysate digest using three different
fragmentation methods. (a) CID, ETD and HCD and (b) CID, ETD and DDDT. All numbers displayed are from single runs.
The method using HCD for fragmentation also
provided more than 1,000 proteins for 1 µg of sample,
demonstrating a much improved success rate of identifying
peptides and proteins compared with the LTQ Orbitrap
XL mass spectrometer, which is also due to an improved
HCD cell resulting in a higher spectral quality.
6
The number of protein IDs with ETD is also very high
yet lower compared to all other methods applied. This was
as expected considering the nature of the sample consisting mainly of smaller peptides resulting in predominantly
doubly charged peptides which provide better fragment ion
spectra quality under CID and HCD conditions.
The location and biological function of all proteins
identified from the proteolytic digest of E. coli whole cell
lysate in this study is detailed in Figure 7.
(a) Molecular Functions
Molecular Functions
 Antioxidant activity
 Catalytic activity
 DNA binding
 Enzyme regulator activity
 Metal ion binding
 Motor activity
9
1021
211
26
382
1
 Nucleotide binding
394
 Protein binding
330
 Receptor activity
 RNA binding
 Signal transducer activity
 Structural molecule activity
 Transcription regulator activity
 Translation regulator activity
16
132
44
72
121
19
 Transporter activity
175
 Unannotated
161
(b) Cellular Component
Cellular Component
 Cell surface
 Chromosome
 Cytoplasm
 Cytoskeletone
 Cytosol
 Endoplasmic reticulum
1
13
657
7
53
6
 Extracellular
43
 Membrane
312
 Mitochondrion23
 Nucleus
 Organelle lumen
 Proteasome
 Ribosome
 Unannotated
32
3
1
67
558
(c) Biological Processes
 Cell communication
Biological Processes
 Cell death
 Cell differentiation
7
8
 Cell division
42
 Cell growth
2
 Cell motility
2
 Cell organization and biogenesis
 Cell proliferation
 Cellular homeostasis
159
3
33
 Coagulation
5
 Conjugation
4
 Defense response
6
 Development
 Metabolic process
 Regulation of biological process
 Reproduction
Figure 7: Gene Ontology annotation of all proteins identified from E. coli digest
by CID, ETD, HCD, and DDDT: (a) molecular functions, (b) cellular component,
(c) biological processes.
87
1
1131
225
20
 Response to stimulus
178
 Transport
254
 Unannotated
205
7
Conclusion
Acknowledgements
The identification of proteins from complex mixtures
requires high sensitivity as well as high acquisition speed.
The technological advancements implemented in the LTQ
Orbitrap Velos mass spectrometer with more than twice
the scan speed of an LTQ Orbitrap XL mass spectrometer,
help to overcome undersampling, increasing the depth of
the analysis of complex proteomes. The enhanced sensitivity and improved duty cycle of the new instrument enables
a dramatic increase of the number of unique peptide IDs,
which translates into an increased number of proteins
identified with higher sequence coverage and confidence.
The LTQ Orbitrap Velos mass spectrometer
We would like to thank Christian Ravnsborg for providing the results from the ProteinCenter™ data interpretation
software and Eduard Denisov for his support and helpful
discussions.
•provides an increased scan speed in MS and MS/MS mode
due to its higher transmission ion source, dual pressure ion
trap and improved HCD cell.
•identifies more than 1,000 proteins per hour from a proteolytic digest of E. coli whole cell lysate.
•provides a higher HCD scan speed and much improved
spectral quality opening up new perspectives for de novo
sequencing, quantitation of chemically labeled samples,
and analysis of modifications which require high mass
accuracy.
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4173-4181.
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References
•offers an increase in experimental throughput, confidently
identifying more peptides and proteins than any other
mass spectrometer currently on the market.
In addition to these
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