Using SILAC and BONCAT to Measure New Protein...
Transcript of Using SILAC and BONCAT to Measure New Protein...
Using SILAC and BONCAT to Measure New Protein Synthesis
Thomas NeubertSkirball Institute
New York University School of Medicine
SILAC and Alternative Labeling Strategies in Quantitative Proteomics Workshop
4:30 PM March 30, 2015
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SILACLabel-Free
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Metabolic Labeling
Fractionation
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LC-MS
Light Heavy
Lysis
MS HL
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Fractionation
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Advantage of SILAC for Comparison of Protein Amounts
David Fenyo NYU School of Medicine
Condition BCondition A
Mix 1:1 Ratio
Tryptic digestion
Quantitation
MS
Identification
MS/MS
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SILAC (Stable Isotope Labeling with Amino acids in Cell culture)
Lys0 Arg0 Lys6 Arg6
SLVGLSQEK
Fractionation or Enrichment (e.g pY IP)
Ong MCP 2002
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Label incorporation through cell division
The Importance of Cellular Context
• Many Receptor Tyrosine Kinases transmit signals through shared pathways but generate distinct biological outputs.
• This may be attributed to cell-specific restriction of responses (available downstream components) or to specific combinations of signals produced by receptor activation.
• Exemplified by differences between cell line data vs. experiments in primary culture or tissue.
Hunter, T. (2000), Cell, 100, 113-127Tan, P.B., et al. (1999), Trends Genet, 15, 145-149
Can we label a differentiated, non-dividing cell population such as primary neurons?
The Key Question for Neuronal SILAC:
There are two important features of cultured dissociated embryonic neurons that givethem promising potential for SILAC experiments.
1. Substantial growth in vitro2. Relatively long period of survival
Label Incorporation in Cultured Neurons
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- Several proteins from whole cell lysates were tracked for label incorporation over a month.
- Results demonstrated that sufficient labeling was achieved to perform SILAC experiments in neurons by 10 days in vitro.
♦ Highest Incorporator● Lowest Incorporator■ Average Incorporation
Sample A, Protein X(Light)
Sample B, Protein X(Heavy)
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Why use a Labeling Efficiency Control?
Multiplex SILAC (Guoan Zhang)
Multiplex SILACSILAC
Label incorporation time course
Lys4 Arg6
Lys8 Arg10
Days in vitro
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Questions:
1. Is it possible to do silac after a brief period of labeling?
2. Is quantitation affected by labeling time?
SILAC incorporation/quantitation
LLASLVK from IPI00763269 Trim28
2 Days in Vitro
6 Days in Vitro
10 Days in Vitro
% Label incorporation ratio
Ratios do not depend on degree of label incorporation
Zhang JPR 2011
NT3 stimulation
Lys8 Arg10Lys4 Arg6
Multiplex SILAC for NT3 signaling in primary (non-dividing) neurons
SDS-PAGE
pY IP
LC-MS/MS
m/z
Inte
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Guoan ZhangMoses ChaoKatrin Deinhardt
Replicate 1 vs Replicate 2
Zhang JPR 2011
Trk pathway
Proteins with ratio changes gene name protein name n ratio (sti/co knownShc3 Isoform p66 of SHC-transforming protein 3 17.2 YesPik3c2b phosphoinositide-3-kinase, class 2, beta polypeptide 13.8 YesNtrk2 Isoform GP145-TrkB of BDNF/NT-3 growth factors receptor precursor 9.8 YesNtrk3 Isoform TRKC of NT-3 growth factor receptor precursor 9.2 YesMapk3 Isoform 1 of Mitogen-activated protein kinase 3 6.8 YesMapk1 Mitogen-activated protein kinase 1 6.3 YesSh2b2 SH2B adapter protein 2 4.4 YesStam signal transducing adaptor molecule (SH3 domain and ITAM motif) 1 4.0 YesPlcg1 148 kDa protein 3.8 YesBdnf Brain-derived neurotrophic factor precursor 3.6 YesScamp3 similar to Secretory carrier-associated membrane protein 3 3.4 NoGrb2 Isoform 1 of Growth factor receptor-bound protein 2 3.0 YesPtpn11 Isoform 1 of Tyrosine-protein phosphatase non-receptor type 11 2.8 YesHgs Isoform 1 of Hepatocyte growth factor-regulated tyrosine kinase substrate 2.8 YesFrs2 fibroblast growth factor receptor substrate 2 2.5 YesPicalm Isoform 2 of Phosphatidylinositol-binding clathrin assembly protein 2.5 YesGrit similar to Rho GTPase-activating protein 2.2 YesCltc Clathrin heavy chain 1 2.0 YesStx12 syntaxin 12 2.0 YesScamp1 Secretory carrier-associated membrane protein 1 1.9 NoClta Isoform Brain of Clathrin light chain A 1.8 YesGria2 Isoform 3 of Glutamate receptor 2 precursor 1.8 YesSez6l2 Isoform 2 of Seizure 6-like protein 2 precursor 1.7 NoVamp2 Vesicle associated membrane protein 2B 1.7 NoRps27a Ribosomal protein S27a 1.6 Yes
13 kDa protein 1.6 YesNsg1 Neuron-specific protein family member 1 1.6 NoNdfip2 Nedd4 family interacting protein 2 1.5 NoVcp Transitional endoplasmic reticulum ATPase 0.7 NoDbnl Isoform 2 of Drebrin-like protein 0.6 NoNckipsd NCK interacting protein with SH3 domain 0.6 NoCapza2;Cav2Hepatocyte growth factor receptor precursor 0.6 No
Zhang JPR 2011
The multiplex partial labeling strategy also can be applied to mice (neucode)
(Don Kirkpatrick, Josh Coon)
Protein Synthesis Changes?
Can we measure BDNF-induced protein translation after 2 hours?
Quantifying Needles in a Haystack
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.-BDNF +BDNF
Solution: Isolate Newly Made Proteins
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.-BDNF +BDNF
Pulsed SILAC: low signal for newly synthesized proteins (short time period)
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A .Normal scheme of peptide elongation
AAA
B. BONCAT
JAAA
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biotin C C
cyclo-addition reaction
biotin
detection on western blots, IP, IF and mass spec
AAAJJ
azide modified polypeptide
alkyne tagged detection molecule
non-canonical amino acid substitutes for methionine-
usually modified to have azide moiety
JAAA
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Developed by David Tirrell and Erin Schuman Labs
Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT)
BONCAT-SILAC workflow to look for BDNF induced proteins*
*First BONCAT-SILAC combination published in Eichelbaum Nature Biotech. 2012
extremely low non-specific binding from BONCAT enrichment
SILAC labeled proteins are highly enriched by BONCAT
BONCAT enhances SILAC signals: critical for quantitation
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pSILAC BONCAT
BONCAT captured nascent proteome is very similar to the stead-state proteome
transcription factors are highly enriched by BONCAT: BONCAT captures short-lived proteins/rapidly induced
proteins
BONCAT finds more BDNF induced proteins than pSILAC
7176 proteins quantified 1840 proteins quantified
Of 53 changing proteins in the BONCAT experiment, 24 are involved in regulation of transcription, including 16 transcription factors
Thanks
Neubert Lab (NYU)Guoan Zhang (SILAC)Dan Spellman (SILAC)
(now at Merck)Steven BlaisJingjing Deng
Fang-ke HuangEsthelle Hoedt
NYUEd Ziff (PSD)
Moses Chao (BDNF)Katrin Deinhardt (BDNF)
(now at U. of Southampton)Heather Bowling (Boncat)Aditi Bhattacharya (Boncat)
Eric Klann(Boncat)David Fenyo (Bioinformatics)
NIH Grants P30 NS050276, S10 RR 017990-01100 Women in Hedge Funds Foundation
Stable Isotope Labeling Approaches in Proteomics: An Open Forum
Chris ColangeloThomas NeubertShao-En OngBrett PhinneyBrian Searle
Stable Isotope Proteomics Open Forum5:30 PM March 30, 2015
Fractionation
Digestion
LC-MS
Lysis
Quantitation – Label-Free (MS)
MS MS
Fractionation
Digestion
LC-MS
Lysis
MS/MSMSMSMS/MS
Quantitation – Label-Free (MS/MS)
HL
Quantitation – Metabolic Labeling
Fractionation
Digestion
LC-MS
Light Heavy
Lysis
MS HL
Fractionation
Digestion
LC-MS
Light HeavyLysis
Quantitation – Protein Labeling
MS HL
Fractionation
Digestion
LC-MS
Lysis
MS
Light
RecombinantChimeric
Proteins (Heavy)
Quantitation – Labeled Chimeric Proteins
HL
Fractionation
Digestion
LC-MS
Light Heavy
Lysis
Quantitation – Peptide Labeling
MS HL
Fractionation
Digestion
LC-MS
Light
Lysis
SyntheticPeptides(Heavy)
Quantitation – Labeled Synthetic Peptides
MS
Fractionation
Digestion
LC-MS
Light Heavy
Lysis
L HMS MS/MS
Quantitation – Isobaric Peptide Labeling
Fractionation
Digestion
LC-MS
Lysis
Quantitation – Label-Free (Standard Curve)
MS
David Fenyo NYU
Advantage of SILAC for Relative Quantitation