proteomic analysis of ribosome heterogeneity proteomics group meeting april 1, 2010 namrata udeshi,...
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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)