Thermo Scientific Velos Pro

Ion Trap LC-MSn

Dual-Pressure Linear Ion Trap

Accelerating Innovation
Designed for quantitation • UHPLC data rates
• Diversity in dissociation • Fast and sensitive MSn
 Accelerating
Innovation
The Thermo Scientific Velos Pro is the pinnacle of ion trap mass spectrometry

that delivers enhanced performance for complex sample analyses. A novel wide

dynamic range discrete dynode detection system offers the ultimate in identification

and quantitation of low-abundance compounds and provides absolute confidence

in every result. For the first time ever with an ion trap, the Velos Pro™ offers Trap-

HCD (Higher-Energy Collisional Dissociation) combined with CID (Collision-Induced

Dissociation), PQD (Pulsed-Q Dissociation) and ETD (Electron Transfer Dissociation)

to solve the most difficult analytical problems. With the improved robustness of

generation II/G2 ion optics, the Velos Pro delivers reliability on the fastest, most-

sensitive, highest capability ion trap available today. The Velos Pro can also be

enhanced with the ultra-high resolution and mass accuracy of Thermo Scientific

Orbitrap Velos Pro or Orbitrap Elite™ technology.

|
2
 Freedom of
Dissociation
The Velos Pro takes ion trap technology to new heights: with CID, PQD, ETD, and now Trap-HCD
available at any level of MS n, the Velos Pro is the most versatile MS instrument on the market.

Trap-HCD of Caffeine

40 60 80 100 120 140 160 180 200 220

m/z

CID of Caffeine

40 60 80 100 120 140 160 180 200 220

m/z

Triple quadrupole-like fragmentation

with no precursor dependent low mass
cutoff is now available on the Velos Pro.
Precursor ions collide with N2 in a high
pressure octopole where collision energy
can be tuned to maximize specific fragments
at any mass-to-charge relative to parent. Collision
energy is normalized for mass-to-charge and for
charge state to deliver optimum fragmentation for all
precursors across the entire mass range. Trap-HCD of small molecules furnishes higher energy transitions;
trap-HCD of peptides offers strong y- and b-ion series as well as highly specific immonium ions.
With CID, PQD, and ETD, Trap-HCD adds capability and flexibility to MS n on the Velos Pro.

|
3
 Amazing Sensitivity –
Maximum Robustness
The Velos Pro presents the latest advancements in ion optics design: the S-Lens delivers dramatically
higher ion currents, and G2 Ion Optics provide a novel mechanism of blocking neutrals outside the
path of the ion beam. These features in combination provide maximum uptime and total confidence
for the most challenging applications.

50 pg on column of Alprazolam and internal

standard in singly crashed plasma
2.000
RSD: 2.07%
Injections: >1300
1.500
Area Ratio

1.000
Rapid: 66.6 kDa/s
3,500 M/∆M
0.500

0.000
0 200 400 600 800 1000 1200 1400
Injection Number
1818 1820 1822 1824 1826 1828

Enhanced: 10 kDa/s
7,600 M/∆M

1818 1820 1822 1824 1826 1828

Ultra Zoom: 27.7 Da/s

36,440 M/∆M

1818 1820 1822 1824 1826 1828

Speed and
Selectivity
The revolutionary Velos Pro is an optimized dual-pressure ion trap with a high pressure
and a low pressure cell. The high pressure cell offers maximum ion trapping, fragmentation,
and isolation efficiency. The low pressure cells enable better resolution and higher scan
rates than any other ion trap mass spectrometer. This dual-pressure trap technology in

|
Velos Pro offers a scan speed for every application: from fast survey scans in the new
4
Rapid Scan mode to maximum resolution in Ultra ZoomScan mode.
 Quantitate Like
Never Before
The Velos Pro blurs the lines between traditional ion trap and triple quadrupole performance. The first trap
of the dual-pressure cell is designed to act like a single quadrupole during the ion loading process,
removing chemical background and minimizing space charge.
Once trapped, precursor ions are fragmented and
detected by a custom-designed

high-performance discrete dynode system optimized to capture the ultra-high

flux of ions ejected from the Velos Pro. The quadrupole-like isolation combined
with high data rate detection produces results only possible on the Velos Pro…
designed with quantitation in mind.

Alprazolam
70000000 Y = 6852.68*X R^2 = 0.9918 W: 1/X
Isolation efficiency of the Velos Pro during the
ion loading process
60000000
Peak Area for 2 Extracted Ions

RT : 0.25
SN : 4
100 100 m/z 524
50000000 Quantitation of Aprazolam from 90
10fg-10ng on column 80
Isolation Efficiency

80
10fg of Alprazolam
40000000 70
on column 60
Relative Abundance

60

50
30000000
40
40

30
20000000 20
20

10
0
10000000
0
0.0 0.2 0.4 0.6 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8
Time (min) m/z at Isolation q
0

0 2000 4000 6000

pg on Column
8000 10000
| 5
 Maximum Information from Proteo
For All Levels of
Maximum Identifications

Routine analysis of complex proteomics samples

is often confounded by the inability to adequately
detect low-abundance proteins, by the large
number of PTMs (Post-Translational Modifications)
possible, and because of the dynamic nature of
the proteome. Ion trap-based mass spectrometry
has demonstrated a superior ability to rapidly
identify and characterize low-abundance proteins
and determine their relative expression levels. It
has become an essential tool in proteomics labs.

The Velos Pro enables a new level of proteomics

sample interrogation, made possible by improve-
Venn diagram from Thermo Scientific Proteome Discoverer 1.3 software showing C. elegans
ments in acquisition rates, MSn sensitivity, selec-
peptide identifications by HCD and CID.
tive accumulation of low-abundance ions and the
addition of a novel fragmentation methodology:
Higher-Energy Collisional Dissociation (HCD). HCD
allows access to low mass fragments such as im-
monium ions which increases the 432.92

confidence in and total number of peptide 3+ 648.88

identifications. 433.24
2+
649.36

Maximum Coverage 100 433.60

432.92

649.88
80
Achieving complete coverage for the purpose of 433.92
Relative Abundance

434.24 650.36
60
sequencing a protein, including its site-specific 432 433 m/z 434 435 648.40
650.88

post-translational modifications, requires 40 648 649 m/z 650 651

648.88
maximizing the number of peptides detected 413.32
20 239.20 269.04
in a single experiment. The application of multiple 105.04 163.04191.04 301.20 359.28 441.32 513.32
659.84
692.84
597.04
0
activation methods, i.e. Collision-Induced 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Dissociation (CID), Electron Transfer Dissociation m/z

(ETD), and now the new Higher-Energy Collisional

Dissociation (HCD) achieves this goal.
HCD of DR VYIHPFHL 2+ ETD of DR VYIHPFHL 3+
An all ion trap-based HCD/ETD decision tree
649.08 649.58
is now possible on the Velos Pro providing 100 100

maximum coverage and characterization 80 80

of target proteins.
Relative Abundance

Relative Abundance

433.17
60 60

40 40
784.58 1298.92
269.33
20 569.50 1028.75 20 592.00
392.92 1000.75 289.33 910.67 1009.75
110.25 1238.83
253.33
0 0
200 400 600 800 1000 1200 200 400 600 800 1000 1200
m/z m/z

6
| Full Scan MS followed by HCD for doubly charged precursor (bottom, left) and ETD for triplycharged
precursor (bottom, right).
omics samples...
Every Target b1

b1
3

3
b1

b1
2

2
a1

a1
6

MS2
b1 4 b1 4

1054.92+2
MS2 b1 3 b1 2
a1

6
b1 4 b1 4
3

Maximum Structural Characterization b1 3 b1 2

a1

925.42+2 MS3
MS3
a1

Mass spectrometry has emerged as one of the most powerful tools

b1 4 b1 4
3

a1
b1 3 b1 2

for the structural elucidation of glycans due to its sensitivity and its 795.84+2 MS4
MS4
ability to analyze complex mixtures. Ion trap mass spectrometry offers a1

multistage fragmentation (MSn ) which is a critical tool for glycan structure

6
b1 4 b1 4
3

a1
MSn spectra and block
elucidation as it allows for determination of structural heterogeneity and 1274.92 +1

MS5
MS5 structure for 9 unit
differentiation of isomers. It is commonly necessary to acquire six or glycan determined by
seven levels of fragmentation (MS6 or MS7) to differentiate potential 3
b1 4 b1 4 LC-MSn on the Velos Pro.
1070.84+1 a1

glycan structural isomers. MS6

MS6

b1 4 b1 4

Relative Quantitation by TMT Labeling

Tandem Mass Tag (TMT) and other isobaric tagging technologies are based on
Results of Trap-HCD TMT Experiments
the release of low-mass reporter ions during fragmentation, allowing simultaneous
identification and quantitation of peptide ions. Both MS3 and Pulsed-Q Dissociation
Peptide Spectral Matches 4363
(PQD) methods are well established linear ion trap applications of these technologies.
The Velos Pro linear ion trap mass spectrometer features a new option. Higher-Energy Peptides Quantified 3269
Collision Induced Dissociation which utilizes a high-pressure RF-only octopole. HCD Percent Quantifiable 75%
is a triple quadrupole-like fragmentation method which produces high intensity reporter Average Protein Level CVs 2.5%
ions resulting in enhanced accuracy and precise quantitation.
Average of the coefficients of variation for
each protein in a 12 protein mixture and
number of IDs by trap-HCD fragmentation.
ITMS, HCD, z=+2, Mono m/z=688.02769 Da, MH+=1375.04810 Da, Match Tol.=1.2 Da

140
b 2+, y 6²+ b 4+
120 Y 7+
458.57 642.67
Intensity [counts] (10^3)

100 Y 4+ Y 6+ 1032.69
b 1+
80 b 3+ 733.62 917.68
343.55 Sample HCD MS2 spectrum for TMT 6-plex
60 529.51 labeled protein digest.
40
20
0
200 400 600 800 1000 1200
m/z

Absolute Quantitation Using AQUA™ Peptides 2.00E+08

1.80E+08
Peak area vs amount of
1.60E+08
GPGEDFR injected for 4
Absolute quantitation strategies such as AQUA center 1.40E+08
orders of magnitude change
around MS2 detection of targeted peptides and their in concentration. GPGEDFR
1.20E+08

1.00E+08
was spiked into unfractionated
6.00E+06

analogues labeled with stable isotope amino acids. The

r2 = 0.9995
5.00E+06

8.00E+07 4.00E+06
human cerebral spinal fluid.
Velos Pro dual-pressure linear ion trap with ultra-fast 6.00E+07 3.00E+06

2.00E+06

scanning and multiple fragmentation techniques delivers 4.00E+07

1.00E+06

2.00E+07 0.00E+00

high precision and accurate quantitation even over

|
0 0.05 0.1 0.15 0.2 0.25
pmol GPGEDFR
0.00E+00

multiple orders of magnitude and in complex matrices. 0 2 4 6 8 10 7

pmol GPGEDFR
 Comprehensive Identification and
of Complex Drug M
113.2 CID MS2 Parent
F
+ F

Maximum Productivity 280.2 N

F
Parent
S
N
The characterization of drug 141.2
Intensity

N
metabolites plays a pivotal role
in pharmaceutical discovery and 70.1
F
F
development. Parent drug and N
F
metabolites need to be identified 248.3
S
98.2 308.3 408.4
262.2
in a variety of biological matrices 409.3
84.2 230.2
over a wide range of concentrations 50 100 150 200 250 300 350 400
m/z
for both in vivo and in vitro studies.
Thermo Scientific MetWorks and
Mass Frontier software, when used
CID MS2 Hydroxylated Metabolite F
with the Velos Pro create a robust, + F
113.2 N OH
sensitive, and integrated workflow F

for identification, characterization, 296.3 S

Hydroxylated Metabolite
and quantitation of drugs and their
N
metabolites in biological matrices. N

141.2
Intensity

OH
F
F
70.1 N
F

The power of MSn can be observed with 264.2

S

the fragmentation and interpretation of 98.2 324.3

278.2
Trifluoperazine and its metabolites which 424.3
84.2
include multiple oxidative metabolites.
MS3 data from HCD fragmentation provides 50 100 150 200
m/z
250 300 350 400
useful diagnostic fragments not readily
observed in a CID MS3 scan.

CID MS3 of m/z 296 264.2 Trap-HCD MS3 of m/z 296; 264.2

shows more diagnostic fragments

+.
NH

F
Intensity

Intensity

235.3
263.2 296.3
263.5
276.3
262.1
227.2 276.2 296.2

8
| 100 150
m/z
200 250 300 50 100 150
m/z
200 250 300
 Quantitation
Metabolism Samples
CID MS2 Dextromethorphan Trap-HCD MS2 Dextromethorphan

215.1 213.0572

147.0023
171.0491
Intensity

Intensity
272.1483

213.1
121.0078 198.0466
147.0
81.1293 241.2167

100 150 200 250 50 100 150 200 250

m/z m/z

With different fragmentation techniques available,

researchers can tailor their choice to meet the
demands of the analysis. For the quantitation of
Dextromethorphan, CID generates an MS2 spectrum
Maximum Performance with a single strong ion, ideal for quantitative
sensitivity, while signal intensity following HCD
fragmentation is spread over multiple MS2 ions.
The increased sensitivity of the Velos Pro
allows simultaneous qualitative and
quantitative metabolite analyses to be 4.0E+07

3.5E+07
carried out easily for both in vivo and in
3.0E+07
vitro studies. The faster scan rates of 2.5E+07
Peak Area

the Velos Pro allow the acquisition of more 2.0E+07

MSn spectra in shorter chromatographic 1.5E+07

1.0E+07
runs. With a full range of fragmentation y = 34201x + 289667
R2 = 0.99974
5.0E+06
techniques available for use at all MSn
0.0E+00
0 100 200 300 400 500 600 700 800 900 1000
stages, researchers can construct Concentration (ng/mL plasma)

higher-quality ion trees for qualitative

metabolite analysis and characterization.
The use of Data Dependant acquisition
coupled with Dynamic Exclusion provides
a methodology for the analysis of both
predicted and unexpected metabolites.

| 9
 Thermo Scientific Application-Specific Software
Turning Data Into Information
Xcalibur™ data system ProteinCenter™ software Mass Frontier™ software
Stable operating platform From data sets to meaningful biology Confident path from spectra
Thermo Scientific Xcalibur software is the Thermo Scientific ProteinCenter is a web- to structure
versatile, easy-to-use data system that based data interpretation tool that enables Thermo Scientific Mass Frontier software
controls all Thermo Scientific MS systems. scientists to compare and interpret data allows confident structural elucidation
The home page of the Xcalibur software sets in minutes instead of months. The through chemically intelligent spectral
offers easy navigation through the software enables filtering, clustering and annotation, state-of-the-art fragmentation
process of instrument setup, sequence statistical bioinformatics analysis utilizing prediction, and unparalleled spectral and
setup, and data acquisition. The XReport a regularly updated, consolidated protein fragmentation mechanism knowledge
package simplifies custom reporting sequence database containing >10 million management.
with drag-and-drop functionality. non-redundant proteins.
SIEVE™ software
Proteome Discoverer software Pinpoint software Analysis of differential expression
Mass informatics platform for Accelerating targeted protein based on comparison of LC-MS
protein scientists quantitation in biological research datasets
Thermo Scientific Proteome Discoverer Thermo Scientific Pinpoint software Thermo Scientific SIEVE software
software is a workflow-based proteomics facilitates the transition from early-stage provides label-free, semi-quantitative
data processing software for in-depth biomarker discovery to larger-scale, differential expression analysis of
data mining of complex LC-MS n data quantitative verification of putative proteins, peptides, or metabolites
sets. With the ability to exploit data from biomarkers and general quantitative from the comparison of multiple LC-MS
different dissociation techniques (CID, proteomics. data sets. It is a statistically rigorous
HCD, IRMPD, ETD and ECD), Proteome tool for analyzing data from metabolism
Discoverer software provides extra MetWorks™ software or biomarker discovery experiments.
certainty for peptide and protein Simplify the interpretation of complex
identifications. Optional inclusion of metabolism data
multiple search algorithms increases Thermo Scientific MetWorks software
analytical flexibility, and results can simultaneously searches multiple
now be merged into a single report modifications of one or more parent
for easier interpretation. drugs and interprets simple to complex
isotope patterns, or unexpected or
low-abundance metabolites.

Thermo Fisher Scientific,

San Jose, CA USA is ISO Certified.

www.thermoscientific.com/velospro
©2011 Thermo Fisher Scientific Inc. All rights reserved. TMT is a registered trademark of Proteome Sciences plc. Mass Frontier is a trademark of by HighChem Ltd.
ProSightPC is a trademark of the University of Illinois at Urbana-Champaign. AQUA™ Peptide is a trademark of Sigma. All other trademarks are the property of
Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please
consult your local sales representative for details.

Africa-Other +27 11 570 1840 Europe-Other +43 1 333 50 34 0 Japan +81 45 453 9100 Spain +34 914 845 965
Australia +61 3 9757 4300 Finland / Norway / Sweden Latin America +1 561 688 8700 Switzerland +41 21 694 71 11
Austria +43 1 333 50 34 0 +46 8 556 468 00 Middle East +43 1 333 50 34 0 UK +44 1442 233555
Belgium +32 53 73 42 41 France +33 1 60 92 48 00 Netherlands +31 76 579 55 55 USA +1 800 532 4752
Canada +1 800 530 8447 Germany +49 6103 408 1014 New Zealand +64 9 980 6700
China +86 10 8419 3588 India +91 22 6742 9434 Russia/CIS +43 1 333 50 34 0
Denmark +45 70 23 62 60 Italy +39 02 950 591 South Africa +27 11 570 1840

BR63415_E 05/11S