Proteomic Analysis of Ribosome Heterogeneity

Proteomics Group Meeting April 1, 2010

Namrata Udeshi, PavanVaidyanathan, Jacob Jaffe, Karl Clauser, Steve Carr, and Wendy Gilbert

Background

Ribosomes are components of cells that make proteins from amino acids.65% ribosomal RNA (rRNA) 35% ribosomal proteins

Eukaryotes have 80S ribosomesTwo subunits small (40S) and large (60S) 79 core ribosomal proteins conserved from yeast to humans

Sources of ribosome heterogeneity translational machinery associated proteins (TMAs) 59 core ribosome proteins in yeast are encoded byduplicated genes ribosomal proteins are post translationally modified

>RPS20: Protein component of the small (40S) ribosomal subunitMSDFQKEKVEEQEQQQQQIIKIRITLTSTKVKQLENVSSNIVKNAEQHNLVKKGPVRLPTKVLKISTRKTPNGEGSKTWETYEMRIHKRYIDLEAPVQIVKRITQITIEPGVDVEVVVASN

Try

psin

Arg

C

Propionylation of epsilon-amino groups on lysines enables ArgC like cleavage by trypsin

Increases hydrophobicity

Utility of using Multiple Enzymes

>RPS21A: Protein component of the small (40S) ribosomal subunitMENDKGQLVELYVPRKCSATNRIIKADDHASVQINVAKVDEEGRAIPGEYVTYALSGYVRSRGESDDSLNRLAQNDGLLKNVWSYSR

Asp

N

LysC

In silico digests with LysC and AspN show production of larger more highly charged peptides

Test a multi-enzyme and multi-activation approach for first steps

Utilize both CAD and ETD

Utility of using Multiple Enzymes

80S

10ug 10ug 10ug

CAD ETD DT CAD ETD DTCAD ETD DT

In-gel* Propionylation + Trypsin

LysC AspN

1ug 1ug 1ug

MS Analysis: LTQ-Velos Orbitrap

each each each

Data Analysis: Spectrum Mill

LC-MS/MS Work Flow

Data Dependent Decision Tree

Procedure controls ETD dissociation based on charge state and mass-to-charge

Procedure:

If the charge state is 3 and the m/z is less than { }, ETD will be performed If the charge state is 4 and the m/z is less than { }, ETD will be performed If the charge state is 5 and the m/z is less than { }, ETD will be performed If the charge state is greater than 5, ETD will always be performed For all other conditions, the currently configured activation type will be performed

Swaney , D.L. et. al. Nature Methods 5, 959 - 964 (2008)

ETD/CAD Exclusive vs. ETD/CAD Decision TreePropionylation

+ Trypsin LysC AspN

Excl

usiv

eD

ecis

ion

Tree

532(88%)

53(9%)

16(3%)

329(60%)

88(17%)

127(23%)

212(78%)

22(10%)

36(12%)

206(80%)

26(10%)

24(10%)

317(64%)

67(14%)

111(22%)

518(78%)

113(17%)

30(5%)

CAD

ETD

Overlap

CAD

ETD

Overlap

Number of unique peptides in an MS analysis (all n=1)

Total=601 Total=544 Total=270

Total=661 Total=495 Total=256

In-solution Digestions with Endoproteinase LysC

27 1718

CAD ETD

Overlap of ribosomal protein identifications using DT

Criteria: >10 % sequence coverage

CAD 27 Ribosomal proteinsETD 17 Ribosomal proteinsOverlap 18 Ribosomal ProteinsTotal 62 Ribosomal Proteins

Protein CAD_PC_>10% ETD_PC_>10% RPL1B CAD ETD RPL38 CAD RPL4A CAD RPL5 CAD

RPL6A ETD RPL8A ETD RPL8B ETD RPL9B ETD RPL10 ETD

RPL11B CAD RPL12B ETD RPL13A CAD RPL13B CAD RPL16A ETD RPL16B CAD ETD RPL17B CAD RPL18B ETD RPL20A CAD RPL21B ETD RPL23A CAD RPL24A CAD ETD RPL24B CAD ETD RPL25 ETD

RPL26B CAD ETD RPL28 ETD RPL30 CAD

RPL33A ETD RPL33B CAD ETD RPL34A CAD RPL34B CAD RPL37A CAD

Protein CAD_PC_>10% ETD_PC_>10% RPS0A CAD RPS0B CAD RPS1A CAD RPS1B CAD ETD RPS2 CAD ETD

RPS4B ETD RPS5 CAD

RPS6B CAD RPS7A CAD RPS7B CAD ETD RPS8A CAD RPS9A CAD RPS9B CAD

RPS10A CAD ETD RPS11B CAD RPS12 CAD ETD RPS15 CAD

RPS17B CAD ETD RPS20 CAD ETD

RPS21B ETD RPS22A RPS23A CAD RPS25A CAD RPS25B CAD RPS26A ETD RPS27A ETD RPS29A CAD RPS29B ETD RPS30A CAD ETD RPS31 CAD ETD

Propionylation + Trypsin & LysC Overlap

Propionylation + Trypsin

LysC

Overlap

13%

23%

Overlap of ribosomal protein identifications using Prop.+ Trypsin & LysC

Criteria: >20 % sequence coverage

64 %

Prop.Tryp 13 Ribosomal ProteinsLysC 23 Ribosomal ProteinsOverlap 64 Ribosomal ProteinsTotal 100 Ribosomal Proteins

493 unique

peptides

390unique

peptides

425 CAD67 ETD

231 CAD159 ETD

Data Dependent Decision Tree

Swaney , D.L. et. al. Nature Methods 5, 959 - 964 (2008)

The probability of either an ETD or CAD MS/MS event resulting in a high-confidence sequence identification

ETD Implemented on an LTQ-Orbitrap MS

McAlister , G.C. et. al. Journal of Proteome Research 7, 3127 - 3136 (2008)

Syka, J.E.P. et al (26) PNAS 2004

Hao, Z., Schwartz, J.C., Humer, A. F.-Poster Thermo Websitehttp://www.sorvall.com/eThermo/CMA/PDFs/Various/File_52633.pdf

ETD Implemented on a Dual-Pressure Linear Ion Trap