1. Proteomic Analysis of Ribosome Heterogeneity Proteomics Group Meeting April 1, 2010 Namrata Udeshi, PavanVaidyanathan, Jacob Jaffe, Karl Clauser, Steve Carr, and Wendy Gilbert

2. Background  Ribosomes are components of cells that make proteins from amino acids.  65% ribosomal RNA (rRNA)  35% ribosomal proteins  Eukaryotes have 80S ribosomes  Two 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 by duplicated genes  ribosomal proteins are post translationally modified

3. >RPS20: Protein component of the small (40S) ribosomal subunit MSDFQKEKVEEQEQQQQQIIKIRITLTSTKVKQLENVSSNIVKNAEQHNLVKKGPVRLPT KVLKISTRKTPNGEGSKTWETYEMRIHKRYIDLEAPVQIVKRITQITIEPGVDVEVVVAS N Trypsin ArgC  Propionylation of epsilon-amino groups on lysines enables ArgC like cleavage by trypsin  Increases hydrophobicity Utility of using Multiple Enzymes

4. >RPS21A: Protein component of the small (40S) ribosomal subunit MENDKGQLVELYVPRKCSATNRIIKADDHASVQINVAKVDEEGRAIPGEYVTYALSGYVR SRGESDDSLNRLAQNDGLLKNVWSYSR AspN 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

5. 80S 10ug 10ug 10ug CADETDDTCADETDDTCADETDDT In-gel* Propionylation + Trypsin LysCAspN 1ug 1ug 1ug MS Analysis: LTQ-Velos Orbitrap each each each Data Analysis: Spectrum Mill LC-MS/MS Work Flow

6. 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)

7. ETD/CAD Exclusive vs. ETD/CAD Decision Tree Propionylation + Trypsin LysCAspN Exclusive Decision 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=601Total=544Total=270 Total=661Total=495Total=256

8. In-solution Digestions with Endoproteinase LysC 27 17 18 CADETD  Overlap of ribosomal protein identifications using DT  Criteria: >10 % sequence coverage CAD 27 Ribosomal proteins ETD 17 Ribosomal proteins Overlap 18 Ribosomal Proteins Total 62 Ribosomal Proteins

9. 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 Proteins LysC 23 Ribosomal Proteins Overlap 64 Ribosomal Proteins Total 100 Ribosomal Proteins 493 unique peptides 390 unique peptides 425 CAD 67 ETD 231 CAD 159 ETD

10. 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

11. ETD Implemented on an LTQ-Orbitrap MS McAlister, G.C. et. al. Journal of Proteome Research 7, 3127 - 3136 (2008)

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

13. Hao, Z., Schwartz, J.C., Humer, A. F.-Poster Thermo Website http://www.sorvall.com/eThermo/CMA/PDFs/Various/File_52633.pdf ETD Implemented on a Dual-Pressure Linear Ion Trap