䮥䭭䮀䬙䭗䭂䮰䮊䮏䬽ེቭ䬺䬙䬠䭚䮆䮺䮘䭶㊂䬽᷹ቯ䎃
䎃
䧊
Ꮉἑᵏม 䎔䫺ኹፒ⌀᮸ 䎔䫺૒⮮ᄥ 䎔䫺⣻Ꮞ䭕䬚䭙 䎔䫺䎶䏗䏄䏓䏈䏏䏖䎃䎰䏄䏕䏗䏋䏄䎃䎧䎑䎕䫺䎯䏄䏑䏊䏕䏌䏇䏊䏈䎃䎭䏄䏐䏈䏖䎃䎬䎑䎖䫺䎳䏌䏓䏈䏕䎃䎦䏋䏈䏏䏖䏈䏄䎗䫺䎽䏋䏒䏘䎃䎤䏑䎗䎃
ᣣᧄ䭭䭰䯃䮆䯃䮁ᩣᑼળ␠ 䎔䫺䎺䏄䏗䏈䏕䏖䎃䎦䏒䏕䏓䏒䏕䏄䏗䏌䏒䏑䎏䎃䎰䏌䏏䏉䏒䏕䏇䎏䎃䎰䎤䎕䫺䎺䏄䏗䏈䏕䏖䎃䎦䏒䏕䏓䏒䏕䏄䏗䏌䏒䏑䎏䎃䎰䏄䏑䏆䏋䏈䏖䏗䏈䏕䎏䎃䎸䏑䏌䏗䏈䏇䎃䎮䏌䏑䏊䏇䏒䏐䎖䫺䎯䏈䏊䏄䏆䏜䎃䎵䏈䏖䏈䏄䏕䏆䏋䎏䎃䎳䏒䏕䏗䏏䏄䏑䏇䎏䎃䎲䎵䎖䎃
OVERVIEW
RESULTS
Brains
⋡⊛䎝䎃䮰䮊䮏䬙䭗䭂䮥䭭䮀䬽ฦེቭ䬺䬙䬠䭚䮆䮺䮘䭶㊂䬽᷹ቯ䎃
Hearts
Kidneys
Livers
Spleens
ᣇᴺ䎝䎃䮰䮠䮲䮜䮱䯃䎃LC/MSE ᷹ቯ䬺䭗䭚⛘ኻቯ㊂䎃
⚿ᨐ䎝䎃ฦེቭ䬺․⇣⊛䬙䭗䭂౒ㅢ䬨䭚䮆䮺䮘䭶䭡หቯ䬦ቯ㊂䬦䬮䎃
Figure 4. ⢄⤳䬺䬙䬠䭚䭾䮏䭶䮴䮧䎃䎳䎗䎘䎓 䭡␜䬦䬮
䮙䯃䮏䮥䮊䮞䬽᜛ᄢ࿑
INTRODUCTION
Hemoglobin subunit alpha
Hemoglobin subunit beta
Actin cytoplasmic
Glyceraldehyde 3 phosphate dh
Serum albumin
Hemoglobin subunit beta 2
Malate dehydrogenase
Actin cytoplasmic 2
Malate dehydrogenase cytoplasmic
Alpha globin
ATP synthase subunit beta
Tubulin alpha 4A chain
Actin alpha skeletal muscle
Peptidyl prolyl cis trans isomerase A
ATP synthase subunit alpha
Alpha enolase
Calmodulin
L lactate dehydrogenase B chain
Tubulin alpha 1C chain
Isocitrate dehydrogenase NADP
Profilin 1
Fructose bisphosphate aldolase A
Superoxide dismutase Cu Zn
Beta glo
Triosephosphate isomerase
⇣䬹䭚∔ᖚ⁁ᘒ䬺䬙䬠䭚䮞䮴䮍䭱䯃䮧⸃ᨆ䭡ⴕ䬕೨䬺䫺䮠䯃䮀䬷䬹䭚ᱜᏱ⁁ᘒ
䬺䬙䬠䭚䮆䮺䮘䭶㊂䬽᷹ቯ䬛ᔅⷐ䬶䬑䭚䫻䬢䭛䭘䬽᷹ቯ䬶䬾䫺䭼䮺䮞䮲䬽೨ಣℂ
䬺䭗䬲䬵↢䬧䭚ᄌേ䭑䬑䭚䬛䫺⇣䬹䭚୘૕䬚䭘↢䬧䭚ᄌേ䭡⍮䭚䬢䬷䬛䬶䬜䭚䫻
䬬䬢䬶ᚒ䫽䬾 䎗 ඘䬽䮰䮊䮏䬙䭗䭂 䎖 ඘䬽䮥䭭䮀䬽 䎘 ⒳㘃䬽ེቭ䯴⣖䫺ᔃ⤳䫺䎃
⣢⤳䫺⢄⤳䫺⣛⤳䯵䬺䬙䬠䭚䮆䮺䮘䭶㊂䭡䫺ᒝᐲ䬽㜞䬓਄૏ 䎖 ୘䬽䮡䮞䮈䮐䎃
䬺䬙䬠䭚䮛䯃䭶㕙Ⓧ୯䭡ౝᮡ䬷Ყセ䬨䭚䬢䬷䬺䭗䭙▚಴䬦䬮䫻I
Figure 2. 䮰䮊䮏⹜ᢱ䬺䬙䬓䬵หቯ䬤䭛䬮 䎔䎕䎕䎖 ୘䬽䮆䮺䮘䭶㊂䭡 䏏䏒䏊䎃䏉䏐䏒䏏䎒䏷䏊 䬶␜䬦䬮䮙䯃䮏䮥䮊䮞䎃 䎃 䎃 䎃 䎃
The colors corresponding to decreasing abundance are in the order of red, black,
and green. Four biological replicates of each organ and four technical replicates of
each digest were performed. Proteins had to be identified in at least three of four
technical replicates of one organ type. Two random proteins replicated, for a false
positive rate of 0.16% at the protein level.
Rat 1
Brains
Hearts
Kidneys
Livers
Spleens
Aspartate aminotransferase
Phosphoglycerate kinase 1
Zero beta globin
Heat shock cognate 71 kDa protein
Elongation factor 1 alpha 1
Cofilin 1
Tubulin alpha 3 chain
Tubulin beta 2A chain
Peroxiredoxin 1
Glutamate dehydrogenase 1
Electron transfer flavoprotein subunit alpha
Phosphatidylethanolamine binding protein 1
L lactate dehydrogenase A chain
Hydroxyacyl coenzyme A dehydrogenase
Nucleoside diphosphate kinase B
Pyruvate kinase isozymes M1 M2
Gamma enolase
Tubulin beta 4
14 3 3 protein zeta delta
Aldehyde dehydrogenase
Peroxiredoxin 2
Cytochrome b c1 complex subunit 6
ADP ATP translocase 2
Phosphoglycerate mutase 1
Electron transfer flavoprotein subunit beta
Table 1. ో 䎘 ེቭ䬶หቯ䬤䭛䬮䮆䮺䮘䭶䬽䮏䮊䮞 䎘䎓 䭡 䏉䏐䏒䏏䎒䏷䏊 䬽㒠㗅䬶
␜䬦䬮䮱䮀䮏
Rat 2
A total of 130 proteins were identified in all of the rat organs. Many of these common proteins are the housekeeping enzymes that are present in all tissues.
Rat 3
Rat 4
Figure 5. Hierarchical clustering of mouse organ data.
Figure 1. 䮰䮊䮏䬽⢄⤳䬚䭘᛽಴䬦䬮⹜ᢱ䭡᷹ቯ䬦䬮⚿ᨐ䬽䭶䮴䮥䮏䭷䮰䮧䎃
Four biological replicates are depicted.
Figure 5. 䮥䭭䮀ེቭ䬽᷹ቯ⚿ᨐ䬺䬙䬠䭚㓏ጀ⊛䭶䮰䮀䮆䮱䮺䭷䎃
Despite the fact that only one technical replicate was performed,
the data from the same organs clustered correctly.
Figure 3. 䮥䭭䮀⹜ᢱ䬽੍஻ಽᨆ䬺䬙䬓䬵หቯ䬤䭛䬮 䎖䎛䎖 ୘䬽䮆䮺䮘䭶㊂䭡 䏏䏒䏊䎃䏉䏐䏒䏏䎒䏷䏊 䬶␜䬦䬮䮙䯃䮏䮥䮊䮞
Three biological replicates of each organ were analyzed once.
METHODS
LC/MS Systems: 1D and 2D nanoACQUITY UPLC®/ SYNAPT™
HDMS™
1D LC System:
In 1D and 2D
Column: 75 μm x 15 cm BEH C18 (1.7 μm)
Gradient: 5-40% B for 90 min at 300 nL/min
100
log fmol/μg
Eluent A: 0.1% formic acid in water
Eluent B: 0.1% formic acid in acetonitrile
2D LC System:
10
In 2D only
First Dimension:
Column: 300 μm x 5 cm XBridge
™
C18 (5 μm)
1
Gradient: discontinuous step gradient at 2 μL/min
Eluent A: 20 mM ammonium formate pH 10.0
0
Eluent B: acetonitrile
Online dilution flow rate: 20 μL/min aqueous
200
400
Protein
600
800
Figure 9: 䎔䎧 䬙䭗䭂 䎕䎧 䭶䮴䮥䮏䭷䮰䮜䭪䬺䭗䭚䮰䮊䮏⣖⹜ᢱ䬽⛘ኻቯ㊂⚿ᨐ
The higher abundance proteins were identified by both
methods.
Figure 6. 䮰䮊䮏⹜ᢱ᷹ቯ⚿ᨐ䬽㓏ጀ⊛䭶䮰䮀䮆䮱䮺䭷
Second Dimension:
Column: 75 μm x 15 cm BEH C18 (1.7 μm)
Gradient: 5-40% B for 90 min at 300 nL/min
45% ACN
Eluent A: 0.1% formic acid in water
CONCLUSIONS
Eluent B: 0.1% formic acid in acetonitrile
20.8% ACN
Sample Preparation:
using
RapiGest
measure
Proteins extracted from rat organs
surfactant.
protein
Bradford
concentration
assay
prior
to
was
used
reduction
x 䮰䮊䮏䬽ེቭ䬚䭘 䎔䎕䎕䎓 એ਄䬽䮆䮺䮘䭶䬛㜞䬓ౣ⃻ᕈ䬶หቯ䫺ቯ㊂䬤䭛䬮
to
17.4% ACN
with
dithiothreitol, alkylation with iodoacetamide, and in-solution
14.5% ACN
x ో 䎘 ེቭ䬺䬙䬓䬵 䎔䎖䎓 ୘䬽౒ㅢ䬨䭚䮆䮺䮘䭶䬛ᬌ಴䬤䭛䬮
digestion with trypsin. Yeast ADH (P00330) was added as an
internal standard for absolute quantitation.
11.1% ACN
Online Dilution with RP/RP:
on
the
second
dimension
To maximize sample recovery
trap
column
from
the
organic-containing fractions, an aqueous flow was delivered
Figure 7. 䮰䮊䮏⣖䬚䭘᛽಴䬦䬮⹜ᢱ䭡৻ᰴర⋡䬶 䎘 䮜䮰䭶䭾䮮䮺䬺
ಽ䬠䬵ಽᨆ䬦䬮ੑᰴర⋡䬽䭶䮴䮥䮏䭷䮰䮧
x 䭾䮏䭶䮴䮧䎃䎳䎗䎘䎓 䬽䭗䬕䬹ེቭ․⇣⊛䬹䮆䮺䮘䭶䬛ᄙ䬞หቯ䬤䭛ቯ㊂䬤䭛䬮
x ↢‛ቇ⊛ᄌേ䭑฽䭐䬮䮆䮺䮘䭶㊂䭡ᱜ⏕䬺᷹ቯ䬨䭚䬢䬷䬺䭗䭙↢‛ቇ⊛ᄌൻ䭡ᝒ
䬗䭚䬢䬷䬛น⢻
with the 2nd dimension pump, and mixed with the eluted
fraction prior to trapping.
x
䎕䎧 䭶䮴䮥䮏䭷䮰䮜䭪䭡↪䬓䭚䬢䬷䬶䭗䭙ᓸ㊂䬹䮆䮺䮘䭶䭡หቯ䬨䭚䬢䬷䬛น⢻䬶䬑䭚
MS Data processing: All data was processed with ProteinLynx
Global Server (PLGS 2.4) with IdentityE informatics2. MSE data
from the individual 2D chromatograms were processed
1D LC
383 Proteins
341 Proteins
2D LC
761 Proteins
separately and then merged into one file prior to database
References
searching. A rat or mouse database with an equal number of
random sequences concatenated onto it was used for the
1. Silva JC, Gorenstein MV, Li GZ, Vissers JP, Geromanos SJ. Absolute quantification of
proteins by LCMSE: a virtue of parallel MS acquisition. Mol Cell Proteomics. 5 (2006) 144
2. Li GZ, Vissers JP, Silva JC, Golick D, Gorenstein MV, Geromanos SJ. Database searching and
accounting of multiplexed precursor and product ion spectra from the data independent
analysis of simple and complex peptide mixtures. Proteomics 9 (2009) 1696
searches to limit the false positive rate to 4%. Hierarchical
clustering was performed in Spotfire.
Figure 8. 䎔䎧 䬙䭗䭂 䎕䎧 䭶䮴䮥䮏䭷䮰䮜䭪䬺䭗䬲䬵หቯ䬤䭛䬮䮆䮺䮘䭶䬽㊀䬹䭙
䭡␜䬦䬮䮠䮺䮇䭫䭩䭷䮰䮧䎃
89% of the proteins identified by 1D were also found in
the 2D analysis.
Acknowledgements
The authors would like to thank Dr. Keith Fadgen and Dr. Scott Geromanos for
their assistance with this work.

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