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

-1 -0.5 0 0.5 1log2(ratio)

SILACLabel-Free

A

HL

Metabolic Labeling

Fractionation

Digestion

LC-MS

Light Heavy

Lysis

MS HL

Fractionation

Digestion

LC-MS

Light Heavy

Lysis

Peptide Labeling

MS

B

Fractionation

Digestion

LC-MS

Lysis

MSMS

Label-Free (MS/MS)C DMetabolic

Label-Free

Log2 ratio-1 -0.5 0 0.5 1

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

XXXXXXXXKXXXXXXXXK

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

1 1:2 1:4 1:8 1:16 1:32 1:64

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

0102030405060708090

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Time In Vitro (days)

% L

abel

Inco

rpor

atio

n

- 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)

m/z

%

0

100

m/z

%

0

100

m/z

%

0

100

m/z

%

0

100

Why use a Labeling Efficiency Control?

Multiplex SILAC (Guoan Zhang)

Multiplex SILACSILAC

Label incorporation time course

Lys4 Arg6

Lys8 Arg10

Days in vitro

0 2 6 10

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

nsity

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

.

. .. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

.

.. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.. .

.

.

.

. .

.

. ..

. ..

. ..

. .

.

. .

.

. .

.

. ..

. ..

. .

.

. .. .

.

. .

.

. .

.

. .

.

. .. .

.

.

.. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.. .

.

. .

.

. .

.

. ..

. ..

. ..

. .

.

. .

.

. .

.

. ..

. ..

. .

.-BDNF +BDNF

Solution: Isolate Newly Made Proteins

.

. .. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

.

.. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.. .

.

.

.

. .

.

. ..

. ..

. ..

. .

.

. .

.

. .

.

. ..

. ..

. .

.

. .. .

.

. .

.

. .

.

. .

.

. .. .

.

.

.. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.

. .

.. .

.

. .

.

. .

.

. ..

. ..

. ..

. .

.

. .

.

. .

.

. ..

. ..

. .

.-BDNF +BDNF

Pulsed SILAC: low signal for newly synthesized proteins (short time period)

470 472 474 476 478 480 482 484m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

HM

L

New protein

Old protein

J J

A .Normal scheme of peptide elongation

AAA

B. BONCAT

JAAA

J

N N

+

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

J

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

470 472 474 476 478 480 482 484m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

HM

L

468 470 472 474 476 478 480 482 484m/z

05

101520253035404550

556065707580859095

100

Rel

ativ

e A

bund

ance

HML

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