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Solutions for Protein<br />
Mass Spectrometry<br />
Innovation you can’t afford to miss<br />
Analyze • Detect • Measure • Control TM
• Ultra-Sensitive Protein Analysis<br />
Finnigan LTQ linear ion trap mass spectrometer<br />
• Sheer Analytical Power<br />
Finnigan LTQ FT hybrid mass spectrometer<br />
• Rapid, Confident Protein ID<br />
Finnigan LTQ with vMALDI source and Ettan DIGE<br />
• Ultimate Analytical Flexibility<br />
MDLC LTQ workstation<br />
• Integrated <strong>Proteomics</strong> Workstation<br />
Finnigan ProteomeX LTQ workstation<br />
• Powerful Bio-Software Solutions<br />
2<br />
Including BioWorks software with<br />
SEQUEST ® algorithm and DeNovoX <br />
sequencing software<br />
Thermo is Revolution<br />
With the complete sequencing of the human genome, accomplished in 2000, a significant<br />
milestone in human scientific pursuit was reached. No sooner was this accomplished, however,<br />
than the race to characterize the protein products of all of those newly-discovered genes began.<br />
The concept of the human proteome and the science of proteomics were born.<br />
The characterization of proteins has proven to be a formidable analytical challenge. Proteins in<br />
biological samples are present in vast numbers, at widely varying concentrations, and in myriad<br />
modified forms. Combine this overt complexity with universal organizational demands for<br />
increased throughput and operational efficiency and the challenge can seem insurmountable.<br />
Fortunately, Thermo Electron can help. With an impressive array of innovative new products,<br />
technologies, and integrated solutions, Thermo is redefining the practical limits of what is<br />
possible in proteomics research.
izing <strong>Proteomics</strong><br />
Innovation you can’t afford to miss!<br />
3
Ultrasensitive Protein Analysis<br />
Finnigan LTQ Linear Ion Trap Mass Spectrometer<br />
4<br />
The revolutionary Finnigan LTQ<br />
linear ion trap mass spectrometer<br />
has pushed protein detection limits<br />
to unimaginably low levels. The<br />
large-capacity linear ion trap stores<br />
dramatically more ions and uses<br />
dual detectors for 100% detection<br />
efficiency.<br />
Thermo’s patented linear ion trap<br />
provides superior effective trapping<br />
capacity with its segmented design.<br />
Complete with radial ejection and<br />
dual detectors, the Finnigan linear<br />
ion trap is the most efficient trap<br />
available, providing power and<br />
sensitivity for all proteomic<br />
applications.<br />
Extremely rapid scan speeds result<br />
in many more MS/MS and MS n<br />
sequencing attempts per unit<br />
time, resulting in far greater<br />
protein coverage than has ever<br />
been possible before.<br />
Finnigan LTQ<br />
linear ion trap
Sheer Analytical Power<br />
Finnigan LTQ FT Hybrid Mass Spectrometer<br />
47 kDa protein<br />
(+ 31 charge<br />
state)<br />
Relative Abundance<br />
100<br />
95<br />
90<br />
85<br />
80<br />
75<br />
70<br />
65<br />
60<br />
55<br />
50<br />
45<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
antiVFab # 99-127 RT: 9.70-14.13 AV: 29 NL: 9.84E4<br />
T:FTMS + pNSIsid = 10.00 Full ms2 1241.00@0.00 [700.00-1800.00]<br />
Relative Abundance<br />
1241.5134<br />
100<br />
95<br />
1241.4363<br />
90<br />
1241.5903<br />
85<br />
80<br />
1241.3849<br />
75<br />
70<br />
65<br />
1241.6418<br />
60<br />
55<br />
50<br />
1241.3335<br />
1241.6932<br />
45<br />
40<br />
35<br />
1241.2823<br />
1241.7445<br />
30<br />
25<br />
1241.2309<br />
1241.7957<br />
20<br />
15 1240.9230<br />
1241.1798<br />
1241.0515<br />
1241.9234 1242.1024<br />
1242.1541<br />
1242.3078<br />
10<br />
5<br />
0<br />
1242.4613<br />
1240.8 1241.0 1241.2 1241.4 1241.6<br />
m/z<br />
1241.8 1242.0 1242.2 1242.4 1242.6<br />
1241.5517<br />
1210.4679<br />
Remove uncertainty from peptide<br />
and PTM assignments. The<br />
extraordinary new Finnigan LTQ FT<br />
incorporates an FT-ICR detector for<br />
ultra-high resolution (>100,000) and<br />
extremely accurate mass measurements<br />
(
Rapid, Confident Protein Identification<br />
Finnigan LTQ with vMALDI Source and Ettan DIGE<br />
Data Dependent MS/MS<br />
of a protein mixture with<br />
several MS/MS spectra<br />
shown<br />
6<br />
Quickly determine which proteins<br />
are present during rapid screening<br />
of proteomics samples by combining<br />
the speed of MALDI ionization<br />
with the sensitivity and MS/MS<br />
performance of the Finnigan LTQ.<br />
Finnigan vMALDI Ion Source<br />
Achieve confident, high-throughput<br />
protein ID with the new intermediate-pressure<br />
Finnigan vMALDI ion<br />
source for the Finnigan LTQ mass<br />
spectrometer. Quickly generate<br />
SEQUEST-searchable MS/MS<br />
spectra for unambiguous protein<br />
identification based on peptide<br />
sequence, not just peptide molecular<br />
weight. And with unique MS n<br />
capability, even post-translational<br />
modification (PTM) spectra (e.g.<br />
phosphorylation, glycosylation)<br />
are easily obtained.<br />
2D Gels and DIGE Chemistry<br />
Chemistry and hardware must<br />
work together to ensure a seamless<br />
system for proteomic research.<br />
Ettan DIGE (2-D Fluorescence<br />
Difference Gel Electrophoresis)<br />
from GE Healthcare (formerly<br />
Amersham Biosciences) is the<br />
state-of-the-art 2D protein expression<br />
visualization system. The Ettan<br />
DIGE system is perfectly suited to<br />
the new vMALDI LTQ mass spectrometer<br />
workstation for maximum<br />
protein throughput.<br />
Cytochrome-C Lactalbumin BSA<br />
Finnigan vMALDI<br />
source with LTQ<br />
linear ion trap
CCD image of protein digest<br />
with α-cyano matrix<br />
Analysis of a four-peptide mixture,<br />
200 amol each, with α-cyano matrix<br />
The Perfect System for High-<br />
Throughput Protein Analysis<br />
The Finnigan vMALDI ion source is<br />
completely optimized to harness the<br />
incredible sensitivity of the Finnigan<br />
LTQ for high-throughput MALDI-<br />
MS/MS analysis. The Finnigan<br />
vMALDI ion source utilizes:<br />
• Crystal Positioning System (CPS) to automatically target<br />
the optimum sampling pattern<br />
• Automated Spectral Filtering (ASF) for real-time monitoring<br />
of spectral quality<br />
• Automatic Gain Control (AGC )<br />
to determine the optimum<br />
number of laser shots for the<br />
best possible sensitivity<br />
Angiotensin I fragment 1-7<br />
Bradykinin<br />
Angiotensin I<br />
Neurotensin<br />
The system is designed to allow<br />
walk-away automation during data<br />
acquisition and has the intelligence<br />
to change experimental conditions<br />
to provide the best possible outcome:<br />
First, the CPS performs a<br />
digital analysis of the sample spot<br />
image to rapidly identify crystals<br />
with good signal-to-noise potential.<br />
CPS also remembers the ablated<br />
path, so that only fresh crystals are<br />
interrogated. Then, the Finnigan<br />
vMALDI ion source begins sampling,<br />
using ASF for real-time monitoring<br />
of spectral quality. When high<br />
quality spectra are detected, the<br />
Finnigan vMALDI ion source uses<br />
AGC to determine the optimum<br />
Automated sampling pattern<br />
mapped with CPS<br />
MS/MS of 50 fmol myoglobin digest,<br />
precursor is the T2 fragment at m/z 1606.9<br />
number of laser shots to achieve<br />
the highest possible sensitivity<br />
without sacrificing spectral quality.<br />
Data Dependent MS/MS is immediately<br />
performed on all precursor<br />
ions, giving MS/MS spectra to identify<br />
all peptides that are present.<br />
Poor signal: Move to different crystal<br />
CPS<br />
ASF<br />
AGC<br />
Good Signal<br />
Excellent Quality Spectra<br />
Examine Spectra<br />
Optimize Laser<br />
CPS, ASF, and AGC allow automated acquisition<br />
of high quality MS and MS/MS spectra<br />
MS 3 of 50 fmol myoglobin digest,<br />
precursor is at m/z 1192.7<br />
Move to next sample: Repeat process<br />
7
Ultimate Analytical Flexibility<br />
MDLC LTQ Workstation<br />
Analytical precision<br />
demonstrated by<br />
duplicate analyses<br />
with the MDLC LTQ<br />
8<br />
Relative Abundance<br />
The combination of the Ettan MDLC<br />
chromatography system from GE<br />
Healthcare and the Finnigan LTQ<br />
from Thermo creates a powerful<br />
new integrated LC/MS system<br />
with extraordinary flexibility and<br />
innovative applications.<br />
RT: 0.00 - 60.01<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
RT: 0.00 - 60.02<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
Relative Abundance<br />
20<br />
10<br />
0<br />
0.10<br />
518.7<br />
5.06<br />
519.0<br />
7.24<br />
535.8<br />
8.60<br />
535.8<br />
11.36<br />
535.8<br />
13.<strong>08</strong><br />
518.8<br />
The pumps perform high pressure<br />
mixing and the system uses realtime<br />
flow monitoring to assure<br />
absolute gradient reproducibility.<br />
The entire system flowpath is bioinert,<br />
allowing high-yield analysis<br />
of post-translationally modified<br />
17.67<br />
563.4<br />
17.86<br />
465.6<br />
19.80<br />
501.9<br />
19.77<br />
501.9<br />
20.67<br />
636.8<br />
22.68<br />
536.8<br />
22.73<br />
536.8<br />
25.32<br />
748.3<br />
27.67<br />
616.3<br />
29.29<br />
460.6<br />
31.14<br />
9<strong>08</strong>.5<br />
peptides. The system can perform<br />
rapid LC/MS separations using a<br />
single column or high resolution<br />
multidimensional separations,<br />
allowing customization of separations<br />
for optimal detection of<br />
peptides by the Finnigan LTQ.<br />
34.75<br />
557.6<br />
36.62<br />
518.7<br />
40.34<br />
518.8<br />
42.27<br />
518.7<br />
44.78<br />
518.7<br />
48.53<br />
518.8<br />
MDLC LTQ provides<br />
ultimate versatility<br />
29.28<br />
460.6<br />
0.23<br />
519.0<br />
5.13<br />
535.8<br />
5.41<br />
518.7<br />
7.62<br />
519.0<br />
9.92<br />
518.7<br />
12.90<br />
518.8<br />
16.24<br />
597.4<br />
17.70<br />
465.6<br />
21.53<br />
650.4<br />
25.31<br />
748.3<br />
26.72<br />
771.4<br />
31.04<br />
9<strong>08</strong>.5 34.45<br />
557.7<br />
37.05<br />
789.5<br />
40.38<br />
518.7<br />
0 5 10 15 20 25 30<br />
Time (min)<br />
35 40 45 50 55 60<br />
41.65<br />
518.7<br />
45.26<br />
518.8 45.61<br />
518.8<br />
52.64<br />
783.8<br />
53.00<br />
783.8<br />
53.56<br />
727.3<br />
56.58<br />
518.7<br />
0 5 10 15 20 25 30 35 40 45 50 55 60<br />
Time (min)<br />
49.75<br />
518.8<br />
53.01<br />
462.2<br />
55.93<br />
519.1<br />
57.91<br />
519.1
Integrated <strong>Proteomics</strong> Workstation<br />
Finnigan ProteomeX LTQ Workstation<br />
Use turnkey, customized proteomics<br />
application methods to perform<br />
advanced protein analysis.<br />
The Finnigan ProteomeX LTQ workstation<br />
integrates optimized, colorcoded<br />
combinations of columns,<br />
Finnigan ProteomeX LTQ workstation with new Micro AS<br />
autosampler for integrated proteomics analysis<br />
plumbing kits, and methods into<br />
easy-to-use configurations for very<br />
specific analytical objectives:<br />
High-Throughput Protein ID<br />
Maximum Sequence Coverage<br />
Phosphorylation Site Mapping<br />
MudPIT<br />
9
High-Throughput Protein ID<br />
Perform a rapid desalting, concentration, and LC/MS analysis for high-throughput protein ID.<br />
Enzymatic digest<br />
of excised<br />
protein spots<br />
Relative Abundance<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
20<br />
90<br />
0<br />
60<br />
22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0<br />
10<br />
50<br />
80Time<br />
(min)<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
Relative Abundance<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
Relative Abundance<br />
100<br />
90<br />
80<br />
70<br />
100<br />
0<br />
40<br />
70<br />
26.30<br />
22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0<br />
100<br />
30<br />
20<br />
60<br />
50<br />
Time (min)<br />
90 Spot # Base Peak<br />
422.0<br />
26.25<br />
422.0<br />
26.34<br />
421.9<br />
80<br />
10<br />
26.23<br />
421.9<br />
40<br />
26.37<br />
422.1<br />
0<br />
70<br />
26.21<br />
25.0<br />
26.0 27.0 28.0 29.0<br />
23.0 24.0<br />
30<br />
422.0<br />
20<br />
60<br />
Time (min)<br />
26.39<br />
422.0<br />
26.41<br />
50<br />
26.19<br />
422.1 422.0<br />
10<br />
26.43<br />
40<br />
422.0<br />
0<br />
23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0<br />
26.48<br />
30<br />
Time (min)<br />
26.16<br />
422.1 422.1<br />
Rapid desalt and<br />
full mass scan<br />
Relative Abundance<br />
Relative Abundance<br />
20<br />
26.53<br />
421.9<br />
25.56 25.61<br />
523.6 523.6 26.14<br />
27.44<br />
422.0 26.64 27.37 27.3727.44<br />
422.0 738.4 738.3 27.48<br />
422.0<br />
10<br />
27.35 738.327.86 738.327.86<br />
25.52 25.77<br />
26.75 26.7527.35<br />
738.3<br />
767.7<br />
523.5 523.6 422.0 422.0738.3<br />
28.0428.26<br />
28.93<br />
767.8<br />
29.06<br />
840.0 419.3419.2<br />
0<br />
22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0<br />
Time (min)<br />
29.27<br />
419.2<br />
85.73<br />
85.83<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
782.1<br />
882.2<br />
981.3<br />
680.3<br />
567.3<br />
1094.3<br />
1336.4<br />
92.69<br />
10<br />
60.93<br />
0<br />
63.94 67.61 72.07 73.60 76.24<br />
82.06<br />
81.62<br />
80.54<br />
89.36 91.84 92.87<br />
101.23<br />
106.35<br />
104.30<br />
107.37<br />
115.51<br />
118.98<br />
121.94<br />
123.74<br />
126.11<br />
60 70 80 90 100 110 120<br />
96.27<br />
97.27<br />
101.75<br />
87.27 95.48<br />
112.10<br />
111.02<br />
Time (min)<br />
Rapid identification of<br />
proteins from 2D gel<br />
using MS/MS data and<br />
SEQUEST searching<br />
algorithms<br />
500 1000 1500 2000<br />
Maximum Sequence Coverage<br />
and perform a Data Dependent Top Ten MS/MS experiment with the Finnigan LTQ.<br />
10<br />
Base Peak<br />
Automated Data<br />
Dependent MS/MS<br />
with Dynamic<br />
Exclusion for<br />
high sensitivity<br />
A mixture of proteins was digested<br />
with enzyme and analyzed with a<br />
120-minute reversed phase gradient.<br />
The Finnigan LTQ, using a Top Ten<br />
Data Dependent Dynamic Exclusion<br />
experiment, conclusively identified<br />
multiple peptides resulting in significant<br />
coverage for all proteins in<br />
the mixture.<br />
Relative Abundance When maximum protein coverage is important, use longer gradients to achieve better peptide resolution<br />
ProteomeX<br />
Methods<br />
Accession<br />
Number Protein<br />
1 P43652 Afamin precursor (Alpha-albumin) (Alpha-Alb).<br />
2 P05091 ALDEHYDE DEHYDROGENASE, MITOCHONDRIAL PRECU<br />
3 P35221 Alpha-1 catenin (Cadherin-associated protein) (Alpha E-catenin).<br />
4 P05067 ALZHEIMER'S DISEASE AMYLOID A4 PROTEIN PRECUR<br />
5 P51693 AMYLOID-LIKE PROTEIN 1 PRECURSOR (APLP)<br />
6 P20073 ANNEXIN A7 (ANNEXIN VII) (SYNEXIN)<br />
7 Q9BZC7 ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBE<br />
8 P02730 Band 3 anion transport protein (Anion exchange protein 1)<br />
9 P01884 BETA-2-MICROGLOBULIN PRECURSOR<br />
10 P55290 CADHERIN-13 PRECURSOR (TRUNCATED-CADHERIN)<br />
11 O14967 Calmegin precursor<br />
12 P17655 Calpain 2, large [catalytic] subunit precursor (EC 3.4.22.17)<br />
13 Q15699 Cartilage homeoprotein 1<br />
14 Q13033 Cell-cycle autoantigen SG2NA (S/G2 antigen)<br />
15 P54219 Chromaffin granule amine transporter (VAT1).<br />
16 P10645 CHROMOGRANIN A PRECURSOR (CGA) (PITUITARY<br />
17 O95239 CHROMOSOME-ASSOCIATED KINESIN KIF4A (CHRO<br />
18 Q9NYC9 CILIARY DYNEIN HEAVY CHAIN (AXONEMAL DYNE<br />
19 Q12860 Contactin precursor (Glycoprotein gP135).<br />
20 P01034 CYSTATIN C PRECURSOR (NEUROENDOCRINE BASIC<br />
21 P00156 CYTOCHROME B<br />
22 Q16850 CYTOCHROME P450 51 (CYPL1) (P450L1) (STERO<br />
23 P17611 Desmin<br />
24 Q9UBP4 DICKKOPF RELATED PROTEIN-3 PRECURSOR (DKK<br />
25 Q12805 EGF-CONTAINING FIBULIN-LIKE EXTRACELLULAR<br />
26 Q15668 EPIDIDYMAL SECRETORY PROTEIN E1 PRECURSOR<br />
27 P23142 FIBULIN-1 PRECURSOR<br />
28 Q9UEY8 GAMMA ADDUCIN (ADDUCIN-LIKE PROTEIN 70)<br />
29 Q9Y6H8 GAP JUNCTION ALPHA-3 PROTEIN (CONNEXIN 46)<br />
30 Q16478 Glutamate receptor, ionotropic kainate 5 precursor<br />
31 P55318 Hepatocyte nuclear factor 3-gamma (HNF-3G)<br />
32 P18065 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN<br />
33 P24592 Insulin-like grow th factor binding protein<br />
34 Q9UKP45 Kelch-like protein 1<br />
35 P10586 LAR protein precursor (Leukocyte antigen related)<br />
36 P03952 lasma kallikrein precursor (EC 3.4.21.34)<br />
37 P<strong>08</strong>547 LINE-1 reverse transcriptase homolog<br />
38 P07864 L-lactate dehydrogenase C chain (LDH-C) (LD<br />
39 O94759 LONG TRANSIENT RECEPTOR POTENTIAL CHANNEL<br />
40 O00754 Lysosomal alpha-mannosidase precursor (Man<br />
41 P04156 MAJOR PRION PROTEIN PRECURSOR (PRP) (PRP27-<br />
42 P40925 Malate dehydrogenase, cytoplasmic (EC 1.1.1.37).<br />
43 O60476 Mannosyl-oligosaccharide 1,2-alpha-mannos<br />
44 P16035 Metalloproteinase inhibitor 2 precursor<br />
45 P20774 Mimecan precursor (Osteoglycin) (Osteoinductive factor)<br />
46 O95255 Multidrug resistance-associated protein 6<br />
47 Q92859 Neogenin precursor.<br />
48 P13591 NEURAL CELL ADHESION MOLECULE, 140 KDA ISO<br />
49 Q9N0X5 Neural proliferation differentiation and control protein-1 precursor<br />
50 P19022 Neural-cadherin precursor (N-cadherin) (Cadherin-2).<br />
51 P07197 Neurofilament triplet M protein (160 kDa neurofilament protein)<br />
52 O43613 Orexin receptor type 1 (Ox1r) (Hypocretin<br />
53 P20774 OSTEOINDUCTIVE FACTOR PRECURSOR (OIF) (OSTEO<br />
54 P10451 OSTEOPONTIN PRECURSOR (BONE SIALOPROTEIN 1)<br />
55 P55058 PHOSPHOLIPID TRANSFER PROTEIN PRECURSOR (L<br />
56 P43034 PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE<br />
57 P07602 Proactivator polypeptide precursor<br />
58 O6<strong>08</strong>83 Probable rRNA processing protein EBP2<br />
59 Q15113 Procollagen C-proteinase enhancer protein precursor (PCPE)<br />
60 P01210 PROENKEPHALIN A PRECURSOR [CONTAINS: MET-EN<br />
Amount Sequence<br />
Protein Injected Peptides Coverage<br />
BSA 100 amol 2 3.95%<br />
Catalase 1 fmol 13 26.48%<br />
Lactoglobulin 1 fmol 2 17.28%<br />
Carbonic Anhydrase 100 fmol 3 15.44%<br />
Lactoperoxidase 100 fmol 24 44.24%<br />
Carboxypeptidase 1 pmol 21 73.06%
Phosphorylation Site Mapping<br />
Identify sites of protein phosphorylation using chromatography columns that provide a high yield of<br />
phosphopeptides plus Data Dependent Neutral Loss MS 3<br />
scanning with the Finnigan LTQ to selectively<br />
perform MS 3<br />
on phosphopeptides.<br />
Relative Abundance<br />
100<br />
50<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
20 .1 5<br />
22 .6 4<br />
27 .6 3<br />
33 .1 3<br />
28 .2 0<br />
33 .5 9<br />
35 .7 6<br />
37 .3 6<br />
39 .5 2<br />
41 .4 0<br />
MS<br />
MS 2<br />
MS 3<br />
41 .7 1<br />
19 .7 3<br />
0<br />
4. 55 9. 44<br />
15 .4 6 42 .7 6 49 .0 4 57 .3 4<br />
0 10 20 30 40 50 60<br />
100<br />
0<br />
476.27 706.<strong>08</strong><br />
688.29<br />
1031.78<br />
1058.62<br />
982.624<br />
747.31 1013.78<br />
503.37 632.39 876.39<br />
1105.42<br />
390.18<br />
844.91<br />
1375.65<br />
1490.75<br />
1361.58<br />
1619 .71<br />
1619.72<br />
80<br />
964.67<br />
60<br />
747.34<br />
1688.81<br />
632.38<br />
40 328.26<br />
503.42<br />
20<br />
603.30<br />
345.07<br />
827.52<br />
1<strong>08</strong>8.34<br />
859.53<br />
1105.56 1361 .71 1601.85<br />
1332 .52<br />
1233.69<br />
1491.61<br />
1070.57<br />
1199.39<br />
1574.61<br />
1817.71<br />
1800 .74<br />
Time (min) m/z<br />
0<br />
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50<br />
100<br />
50<br />
0<br />
40 m M<br />
150 160<br />
0 10 20 30 40 50 60 70 80<br />
Time (min)<br />
90 100 110 120 130 140<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
80 m M<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
100<br />
50<br />
(Shows<br />
neutral loss)<br />
0 m M<br />
10 m M<br />
20 m M<br />
60 m M<br />
100 m M<br />
120 m M<br />
150 m M<br />
200 m M<br />
400 m M<br />
800 m M<br />
0<br />
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160<br />
Time (min)<br />
Relative Abundance<br />
40 0 60 0 80 0 1000 1200 1400 1600 1800 2000<br />
Analysis of a beta casein digest, showing base peak LC/MS analysis, MS spectrum, MS/MS spectrum showing neutral loss<br />
indicative of a phosphopeptide, MS 3 spectrum showing excellent fragmentation, and definitive identification by SEQUEST.<br />
FQS*EEQQQTEDELQDK<br />
Multidimensional Protein ID (MudPIT)<br />
Analyze complicated protein digest mixtures with automated, on-line multidimensional LC/MS analysis. Peptides<br />
are retained on a strong cation exchange column, desalted and concentrated on peptide traps, then resolved on a<br />
high-performance C18 column and extensively mined for peptides by the Finnigan LTQ. Results from all salt steps<br />
are compiled in a final Multiple File Consensus Report by BioWorks.<br />
Neurofilament triple-m protein (125 ng/L in normal CSF)<br />
present at < 1 fmol in this analysis<br />
Partial list of<br />
proteins found<br />
ProteomeX<br />
Methods<br />
Accession Unique Coverage<br />
Number Protein Peptides Percentage<br />
P02763 ALPHA-1-ACID GLYCOPROTEIN 2 PRECURSOR 5 18<br />
P19652 ALPHA-1-ACID GLYCOPROTEIN 2 PRECURSOR 8 28<br />
P04217 ALPHA-1B-GLYCOPROTEIN 12 25<br />
P01009 Alpha-1-antitrypsin precursor 10 26<br />
P02765 ALPHA-2-HS-GLYCOPROTEIN PRECURSOR 10 31<br />
P01023 ALPHA-2-MACROGLOBULIN PRECURSOR 35 48<br />
P01011 ALPHA-1-ANTICHYMOTRYPSIN PRECURSOR 25 84<br />
P43652 AFAMIN PRECURSOR (ALPHA-ALBUMIN) 3 25<br />
P02768 SERUM ALBUMIN PRECURSOR 67 58<br />
P01876 IG ALPHA-1 CHAIN C REGION 2 25<br />
P01019 ANGIOTENSINOGEN PRECURSOR 11 25<br />
P010<strong>08</strong> ANTITHROMBIN-III PRECURSOR (ATIII) 3 24<br />
P02647 APOLIPOPROTEIN A-I PRECURSOR (APO-AI) 6 16<br />
P02652 APOLIPOPROTEIN A-II PRECURSOR (APO-AII) 5 20<br />
P06727 APOLIPOPROTEIN A-IV PRECURSOR (APO-AIV) 4 15<br />
P06727 APOLIPOPROTEIN A-IV PRECURSOR (APO-AIV) 7 25<br />
P05090 APOLIPOPROTEIN D PRECURSOR 8 49<br />
P02649 APOLIPOPROTEIN E PRECURSOR (APO-E) 11 32<br />
P02749 Beta-2-glycoprotein I precursor (Apolipopro 5 12<br />
P32247 BOMBESIN RECEPTOR SUBTYPE-3 (BRS-3) 5 24<br />
P09871 COMPLEMENT C1S COMPONENT PRECURSOR 2 35<br />
P49454 CENP-F KINETOCHORE PROTEIN 3 45<br />
P00450 CERULOPLASMIN PRECURSOR (FERROXIDASE) 4 25<br />
P00751 COMPLEMENT FACTOR B PRECURSOR 23 37<br />
P<strong>08</strong>603 COMPLEMENT FACTOR H PRECURSOR 15 42<br />
P10909 Clusterin precursor (Complement-associated 15 25<br />
P09871 Complement C1s component precursor 2 24<br />
P01024 COMPLEMENT C3 PRECURSOR 38 37<br />
P01028 COMPLEMENT C4 PRECURSOR 32 23<br />
Q12860 CONTACTIN PRECURSOR (GLYCOPROTEIN GP135) 6 16<br />
P11532 DYSTROPHIN 5 25<br />
P02671 FIBRINOGEN ALPHA/ALPHA-E CHAIN PRECURSOR 13 34<br />
P02751 FIBRONECTIN PRECURSOR (FN) (COLD-INSOLUBLE 3 24<br />
P01857 IG GAMMA-1 CHAIN C REGION 21 60<br />
P01859 IG GAMMA-2 CHAIN C REGION 14 30<br />
P01860 IG GAMMA-3 CHAIN C REGION (HEAVY CHAIN DISEA 13 34<br />
P01861 CHAIN C REGION 3 15<br />
P01777 HAIN V-III REGION TEI 3 14<br />
P01834 HAIN C REGION 3 50<br />
P80362 HAIN V-I REGION WAT 7 21<br />
P18136 HAIN V-III REGION HIC PRECURSOR 6 16<br />
P06396 GELSOLIN PRECURSOR, PLASMA 16 27<br />
P02023 HEMOGLOBIN BETA CHAIN 32 23<br />
P02790 HEMOPEXIN PRECURSOR (BETA-1B-GLYCOPROTEIN) 12 25<br />
P00738 HAPTOGLOBIN-2 PRECURSOR 15 25<br />
P00739 HAPTOGLOBIN-RELATED PROTEIN PRECURSOR 2 24<br />
P01042 Kininogen Alpha-2-thiol proteinase inhibitor 38 37<br />
P01597 KAPPA CHAIN V-I REGION DEE 32 23<br />
P01842 IG LAMBDA CHAIN C REGIONS 3 50<br />
Q92876 KALLIKREIN 6 PRECURSOR (PROTEASE M) (NEURO 2 23<br />
P29622 Kallistatin precursor (Kallikrein inhibitor) 2 25<br />
Q9UM88 Beta 2-microglobulin protein 9 70<br />
P36955 MENT EPITHELIUM-DERIVED FACTOR PRECURSOR 6 23<br />
P05155 PLASMA PROTEASE C1 INHIBITOR PRECURSOR (C1 I 13 34<br />
P00747 PLASMINOGEN PRECURSOR 33 44<br />
P02787 SEROTRANSFERRIN PRECURSOR 34 44<br />
P05452 TETRANECTIN PRECURSOR (TN) (PLASMINOGEN-KRI 6 16<br />
P07477 TRYPSINOGEN I PRECURSOR 15 33<br />
P02766 Transthyretin precursor (Prealbumin) (TBPA) 6 16<br />
P02774 VITAMIN D-BINDING PROTEIN PRECURSOR (DBP) 7 18<br />
P04004 TRONECTIN PRECURSOR (SERUM SPREADING FACTOR) 2 35<br />
11
Powerful Bio-Software Solutions<br />
Achieve Dramatic Productivity Gains<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
100<br />
80<br />
13 .1 9<br />
Efficiently turn data into information,<br />
and then into knowledge, with<br />
Thermo’s solutions:<br />
BioWorks–Protein identification<br />
software, based on SEQUEST<br />
SEQUEST Cluster–Parallel<br />
processing for accelerated database<br />
searching<br />
DeNovoX–Automated de novo<br />
sequencing software<br />
SALSA–Spectral pattern<br />
recognition software<br />
Starting with MudPIT data, BioWorks identifies and highlights the b- and y-ions<br />
matched within the peptide chain of the first identified protein candidate.<br />
12<br />
RT: 0.00 - 147.01<br />
Relative Abundance<br />
25 .6 3<br />
43 .98<br />
43 .1 0<br />
BioWorks Protein<br />
Identification Software<br />
Use the most-cited protein identification<br />
algorithm in the scientific<br />
literature–SEQUEST–to correctly<br />
match MS, MS/MS, and MS n<br />
spectra with protein databases.<br />
Renowned for its sensitivity and its<br />
ability to identify PTMs, the power<br />
of BioWorks is amplified by:<br />
3. 85<br />
45 .49 48 .38<br />
53 .4 2 62 .96 79 .4 8 97 .29 11 1.60 12 1.87<br />
13 0.22<br />
27 .6 7<br />
23 .3 0<br />
43 .0 8<br />
19 .68 39 .16 29 .3 4<br />
18 .10<br />
14 .07 51 .0 5<br />
53 .6 7<br />
61 .2 6 75 .8 2 94 .59 1 04. 07<br />
11 6.91<br />
12 8. 6 6 13 2. 90<br />
13 5. 25<br />
28 .2 3<br />
24 .11<br />
33 .86<br />
23 .2 1 36 .15<br />
16 .50<br />
44 .1 4 57 .31<br />
14 .6 5<br />
10 .12<br />
62 .0 0<br />
75 .8 4<br />
97 .1 4 10 2. 90 12 2. 3 7 13 0. 0 0<br />
38. 40 46 .50<br />
29 .91<br />
57 .2 1<br />
60<br />
25 .0 2<br />
40<br />
20<br />
0<br />
15 .27 19 .7 9<br />
1.30<br />
58. 33<br />
75 .9 5 98 .8 4 101 .2 1<br />
12 4.47 13 2. 44<br />
0 20 40 60 80 100 12 0 14 0<br />
Time (min)<br />
Post-Translational<br />
Modification Searches<br />
Automatically set up and execute<br />
searches for static and dynamic<br />
modifications such as phosphorylation<br />
and oxidation<br />
Protein Quantitation<br />
using XPRESS Automatically generate relative<br />
quantitation levels in peptides<br />
tagged with any label such as<br />
ICAT reagent labeled peptides<br />
Multiple File Consensus<br />
Consolidate and sort results from<br />
a number of SEQUEST searches<br />
–essential for MudPIT analyses<br />
Customized Results<br />
Filtering and Sorting<br />
Incorporate XCorr vs. Charge State<br />
filter to automatically eliminate<br />
low-scoring peptides and proteins,<br />
and reduce the amount of time<br />
spent verifying results<br />
Database Management<br />
Automatically <strong>download</strong> protein<br />
database updates from public web<br />
sites, index them to increase their<br />
specificity, and create customized<br />
databases with proprietary proteins<br />
SALSA<br />
Mine MS/MS spectra for unexpected<br />
post-translational modifications<br />
or mutations with this spectral<br />
matching tool
SEQUEST Cluster<br />
SEQUEST searching speed can<br />
be scaled to the needs of each<br />
individual lab. As overall throughput<br />
and data processing needs<br />
increase, computational capacity<br />
can be easily added. The SEQUEST<br />
Cluster creates a parallel processing<br />
software architecture to distribute<br />
searches across many<br />
individual processors, greatly<br />
reducing search times.<br />
Absolute and relative probabilities of partial and<br />
completed sequences are sorted and displayed<br />
in this colorful results table.<br />
Singly and doubly-charged b- and y-ions are highlighted in the spectral<br />
display, while amino acid positions are annotated individually.<br />
DeNovoX—the “One-Click”<br />
Solution to Automated<br />
de novo Sequencing<br />
Use this software to sequence<br />
a novel protein or confirm the<br />
identity of a protein candidate<br />
from a BioWorks database search.<br />
DeNovoX uses a rigorous, probability-based<br />
algorithm to derive<br />
sequences for peptides from the<br />
information contained in their<br />
MS/MS spectra.<br />
Interactive and intuitive graphics<br />
guide a user through a list of possible<br />
peptide tags, their b- and y-ion<br />
series, and fragment assignments.<br />
Sequence gaps are automatically<br />
revealed to help assign PTMs.<br />
This Cluster has the ability to search 20,000 spectra against<br />
the human database in just a few minutes to dramatically<br />
streamline the protein identification process.<br />
13
Complete <strong>Proteomics</strong> Workflow<br />
Effective Analysis of Proteins<br />
14<br />
Sample Preparation<br />
Effective analysis of proteins<br />
requires a strategy for reduction<br />
of sample complexity using gels,<br />
chromatography, or affinity-based<br />
separations. A strategic <strong>Proteomics</strong><br />
Alliance with GE Healthcare gives<br />
Thermo customers direct access<br />
to GE Healthcare technologies and<br />
support. You can call upon us to<br />
help solve your most challenging<br />
separations needs utilizing Ettan<br />
gels, CyDye reagents, DeCyder <br />
software, and AKTA and Ettan chromatography<br />
systems and supports.<br />
Sample<br />
preparation<br />
Protein Identification<br />
using MALDI<br />
The Finnigan vMALDI source for<br />
the Finnigan LTQ creates a powerful<br />
new system for high-throughput<br />
protein ID using MALDI-MS/MS<br />
capability to provide protein ID<br />
based on peptide structure, not<br />
just peptide MW. This allows rapid<br />
identification of proteins by effective<br />
analysis of protein digest mixtures.<br />
2D gel<br />
electrophoresis<br />
Multidimensional<br />
liquid chromatography
Protein Identification<br />
using Electrospray<br />
The Finnigan LTQ is the ideal MS<br />
system for protein analysis. The<br />
sensitivity and rapid scanning of<br />
the LTQ are combined to provide the<br />
best possible protein coverage and<br />
PTM identification for both simple<br />
and complex samples.<br />
The Finnigan ProteomeX LTQ system<br />
provides an integrated, turnkey<br />
solution for most proteomics applications,<br />
including high throughput<br />
protein ID, phosphorylation site<br />
mapping, and MudPIT.<br />
Protein identification using<br />
mass spectrometry<br />
Where additional capability is<br />
required, the MDLC LTQ system<br />
delivers ultimate versatility and<br />
analytical performance.<br />
FT-MS<br />
The Finnigan LTQ FT Hybrid provides<br />
extraordinary mass accuracy and<br />
resolution for difficult challenges<br />
such as PTM characterization and<br />
complex sample analysis.<br />
Bioinformatics<br />
TurboSEQUEST, incorporated into<br />
BioWorks, is the most cited protein<br />
identification algorithm in the scien-<br />
tific literature, yielding highly<br />
sensitive and confident answers.<br />
Both false positive and false negative<br />
rates for incorrect protein identification<br />
have fallen dramatically,<br />
reducing the burden of manual,<br />
post-analysis data review.<br />
Where increased computational<br />
speed is required, the SEQUEST<br />
Cluster can apply the power of a<br />
supercomputer to the analysis of<br />
your data, scalable to the needs<br />
of your lab.<br />
Bioinformatics<br />
• Identification<br />
• Quantification<br />
• Characterization<br />
15
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