Increasing MS Sensitivity for Proteomics

Transcription

1 Increasing MS Sensitivity for Proteomics Discovery to Pathways Analysis: Using Agilent 6550 ifunnel Q-TOF Vadi Bhat, Ph.D. Senior Applications Scientist Agilent Technologies Wilmington, DE, USA 1 April 2, 2013

2 Improvements for Increasing Protein Identification 2500 LMZ308 LN18 SNB19 U r r r r r r r3 2 April 2, 2013

3 Protein Discovery Workflow Proteases Complex Proteomics standard (Pfu) OFFGEL Columns for depletion and fractionation HPLC-Chip 6550 Q-TOF Spectrum Mill 1290 Infinity UHPLC MPP Bravo 3 April 2, 2013

4 Improvements for Increasing Protein Identification Increased identifications Data dependent acquisition (DDA) Chromatography Q-TOF mass spectrometer Recognize peptide isotope pattern Better phase (Polaris) Increase sensitivity Select pure precursors Improved chip manufacturing Increase speed to acquire more MS/MS Optimize MS/MS accumulation time 4 April 2, 2013

5 HPLC-Chip/MS Interface Robust, Automated Nanospray Reproducibility Chip to Chip Instrument to Instrument Low-Volume Connections High Resolution, High Sensitivity Available on ALL Agilent LC/MS Systems X MRM for Biomarker Validation Page 5 5 April 2, 2013

6 Total ion Chromatogram (TIC) and Base peak Chromatogram (BPC) of Triplicate Analysis of Yeast Digest, 2mg on High Capacity Chip, 60 min Gradient TIC BPC 6 April 2, 2013

7 Improved Chromatography With Polaris C18, 3 µm Stationary Phase Improved Protein ID Zorbax 300SB-C18 Jupiter MagicAQ SB-AQ/Reprosil Extend C18/Reprosil MetaSil Polaris Best of tested phases Narrower peaks than existing HPLC-Chips Good performance with high sample load 7 April 2, 2013

8 HPLC-Chips For Many Applications AVAILABLE NOW! Protein ID Peptide Quantitation Biomarker Discovery & Validation Phosphopeptide Analysis Glycan Analysis Intact Protein Small Molecule Analysis RNA/DNA Analysis Custom Chips for customized success mab GlycoPRO Chip Polaris HR Chip Intact Protein Analysis Quantitative Proteomics Glycoprotein Analysis Phosphoprotein Analysis Peptide/protein digests Biomarker Discovery Interchangeable chips for your workflow of choice Page 8 April 2, 2013

9 G1992A: A Flexible Nanoelectrospray Source For Agilent Mass Spectrometers Easily interfaced to any nanolc system Easily used with offline nanoesi Robust, precise positioning of nano emitter Close source for reduced background noise Emitter position easily visualized on PC with USB camera connection Easy UIRM for TOF/Q-TOF using wick (like HPLC-Chip) 9 April 2, 2013

10 Improvements for Increasing Protein Identification Increased identifications Data dependent acquisition (DDA) Chromatography Q-TOF mass spectrometer Recognize peptide isotope pattern Better phase (Polaris) Increase sensitivity Select pure precursors Improved chip manufacturing Increase speed to acquire more MS/MS Optimize MS/MS accumulation time 10 April 2, 2013

11 Acc. Time (ms) DDA: On-the fly Optimization of MS/MS Acquisition Time Based on Precursor Intensity Power ramp relationship Increased protein identified Increased # of MS/MS Faster cycle times MS precursor abundance 11 April 2, 2013

12 Comparing MS/MS spectra (500ng Vs. 10ng): IVSNASCTTNCLAPLAK: GAPDH 500ng 10ng 12 April 2, 2013

13 Improvements for Increasing Protein Identification Increased identifications Data dependent acquisition (DDA) Chromatography Q-TOF mass spectrometer Recognize peptide isotope pattern Better phase (Polaris) Increase sensitivity Select pure precursors Improved chip manufacturing Increase speed to acquire more MS/MS Optimize MS/MS accumulation time 13 April 2, 2013

14 Agilent 6550 Q-TOF with ifunnel Technologies Longer Invar flight tube (resolution + stable mass accuracy) New- Dual Agilent JetStream orthogonal spray (robust and stable mass calibration) New- ifunnel (10X sensitivity gain) New- Ion Beam Compression and Shaping (resolution + sensitivity) Hexapole axial focusing collision cell (faster MS/MS spectra) New embedded processor (50 spectra/sec) 4 GHz digitizer + ADC detector (resolution, mass accuracy, dynamic range) 14 April 2, 2013

15 ifunnel Technology Enhances Q-TOF Sensitivity By Sampling/Transmitting More Ions Samples more ions Removes neutrals Gives higher ion yield 15 April 8, 2, 2013

16 Overlapped BPC of ifunnel 6550 QTOF and 6520 QTOF data April 2, 2013

17 Few examples of proteins that were identified either unique or with more peptides in 6550 ifunnel QTOF 6550 QTOF #peptides 6520 QTOF # peptides 77 unique 37 unique 43 unique 27 unique 52 unique 9 unique %AA Coverage Score unique 5 unique Protein Name Neuroblast differentiationassociated protein Glyceraldehyde-3-phosphate dehydrogenase Cytoplasmic dynein 1 heavy chain 1 Dolichyldiphosphooligosaccharide--protein glycosyltransferase 17 unique 7 unique Serine hydroxymethyltransferase, mitochondrial 15 unique 1 unique Ribosome-binding protein 1 17 unique 3 unique Exportin-1 14 unique 3 unique kda U5 small nuclear ribonucleoprotein 13 unique 2 unique Far upstream element (FUSE) binding protein 1 15 unique 3 unique Isoform 3 of Importin-5 14 unique 0 unique Coatomer subunit gamma 11 unique 2 unique Inorganic pyrophosphatase 6550 QTOF # peptides 6520 QTOF # peptides %AA Coverag e Score Protein Name 13 unique 2 unique Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 10 unique 5 unique Eukaryotic initiation factor A-III 8 unique 0 unique Kinectin 8 unique 0 unique Vigilin 8 unique 0 unique Nuclear pore complex protein Nup205 9 unique 3 unique Talin-2 7 unique 0 unique Eukaryotic translation initiation factor 2 9 unique 0 unique Glycogen phosphorylase, brain form 8 unique 0 unique N-acylaminoacyl-peptide hydrolase 7 unique 1 unique Non-erythrocytic betaspectrin unique 1 unique EIF4G3 protein 3 unique 0 unique Chromosome 1 open reading frame 129 2mg of LN18 cell lysate was analyzed in triplicates on HPLC-Chip-6550 ifunnel QTOF and 6520 QTOF using 90min gradient (3-40% B) 17 April 2, 2013

18 # of Unique Proteins Proteomics profiling of 4 different types of GBM cell types in 2 different types of Q-TOF s Number of distinct proteins identified in each analysis 2500 LMZ308 LN18 SNB19 U r r r r r r r3 Samples: University of Texas MD Anderson Cancer Center 18 April 2, 2013

19 Venn diagram showing the proteomics comparison of 4 different types of GBM cell types in 2 different types of Q- TOFs, April 2, 2013

20 Spectrum Mill MPP Data Exchange: A Unique Agilent Advantage 20 April 2, 2013

21 PCA analysis and Hierarchical clustering of differential proteins within each cell types MAPKK 1 Nestin LDHA MAP1B 21 April 2, 2013

22 MAP kinase cascade in four GBM cell lines 22 April 2, 2013

23 # of Unique Proteins OFE Fractionation for HeLa Cell Lysate Increased Protein Identification HeLa 2ul 5238 entities HeLa 5ul 6919 entities HeLa 6ul 7552 entities HeLa_2ul HeLa_5ul HeLa_6ul OGE fractionation Samples and OGE Fractionation: Cold Spring Harbor Lab 23 April 2, 2013

24 OFE Fractionation for HeLa Cell Lysate Increased Protein Identification Sample # of Runs MS/MS spectra collected MS/MS spectra merged MS/MS spectra filtered MS/MS spectra valid Validation Yield # Distinct Peptides FDR Protein (%) # Distinct Protein HeLa_2ul HeLa_5ul HeLa_6ul All Sample # of Runs MS/MS spectra collected MS/MS spectra merged MS/MS spectra filtered MS/MS spectra valid Validation Yield # Distinct Peptides FDR Protein (%) # Distinct Protein U2OS-r U2OS-r U2OS-r All April 2, 2013

25 One-Carbon-Metabolism pathway 25 April 2, 2013

26 MAP kinase cascade in HeLa and U2OS cell line 26 April 2, 2013

27 27 April 2, 2013