The Perfinity Workstation: Achieving Quality Through Automation

Transcription

1 The Perfinity Workstation: Achieving Quality Through Automation Scott A. Kuzdzal, Ph.D. Bioprocessing and Process Development Symposium (BPD) "Analytical Technologies" 13 October, 2011

2 Shimadzu History Established in Nijo area of Kyoto's Kiyamachi district Started manufacture and sales of physical and chemical instruments Succeeded in Japan's first manned balloon flight Started production of storage batteries Succeeded in taking radiographs Built Japan's first medical X-ray apparatus Successful balloon flight (1877) Early X-ray radiographs (1896) Delivered X-ray apparatus to Japan Red Cross' Ohtsu hospital (1911) Founder Genzo Shimadzu Genzo Shimadzu Jr. 2

3 Full Line of MS Products LCMS-2020 Single Quad LCMS-8030 Triple Quad LCMS-IT-TOF (Structural & Metabolite ID) GCMS-QP2010 Ultra PERFINITY WORKSTATION (Automated Protein Sample Prep) AXIMA MALDI

4 Shimadzu Platforms & Solutions Shimadzu PLATFORMS provide the greatest versatility and performance Perfinity Workstation Automated Protein Sample Prep MegaTOF Pharmaceutical Aggregates & Ultra High Mass Samples 2D HPLC for Bioanalysis Analyze LMW analytes directly from complex fluids AXIMA id Plus Microorganism ID And much, much more!

5 Protein/Peptide Analysis Challenges Proteins and peptides have a growing impact on the areas of pharmaceutical development and disease diagnostics Samples are extremely complex, containing thousands of proteins with target proteins occurring at trace levels» Like looking for a needle in a haystack 5

6 Protein/Peptide Analysis Challenges Sample purification the time and quality bottleneck Traditional protein sample prep and analysis workflows often take upwards of 72 hours of multiple step processes Each step introduces variability in conversion and recovery Resulting in %CVs often in excess of %» Hardly Quantitative!!! 6

7 Protein/Peptide Analysis Challenges Typical Processes in a Mass Spec Sample Preparation Affinity Selection Buffer exchange Digestion Desalting Reverse phase separation 7

8 Protein/Peptide Analysis Challenges Typical Processes in a Mass Spec Sample Preparation Affinity Selection Buffer exchange Digestion Desalting Reverse phase separation 8

9 Protein Challenges: Digestion uv minutes 1 hour 3 hours 6 hours 12 hours 18 hours 24 hours GFFYPTK Protein Product Undigested Protein min Digestion of Insulin Under Standard Conditions The Traditional (solution) Way 9 Sample: 5ug insulin Column : HALO 2.1x100mm RPC Mobile Phase A: 2% ACN, 98% water, 0.1% Formic Acid Mobile Phase B: 90% ACN, 10% water, 0.1% Formic Acid Detection: UV/VIS at 214nm

10 Proteomics Challenges: Digestion How does this affect your results? Couldn t sleep Slept in 10

11 Perfinity Workstation what is it? Automated solution for targeted proteomics Affinity-based capture of target proteins and online digestion and reversed phase separation of peptides Can be also be used without affinity capture step i.e. automated, reproducible digestion & peptide separation only 11

12 Perfinity Workstation Based on the Shimadzu modular HPLC system System controller Ovens, switching valves and columns UFLC Autosampler LC gradient pumps UFLC UFLC 12 UV detector Buffers pump

13 Perfinity Workstation Pump Waste Trypsin Buffer Exchange Waste Blank Waste Affinity Valve 1 Valve 2 Valve UV/Vis RPC Perfinity Workstation Plumbed for - Affinity Selection Buffer Exchange Trypsin Digestion Desalting Reverse Phase Separation Waste Outlet to MS or Waste Waste Pump Desalt Pump

14 Perfinity Workstation: 4 min Digests!!! uv Data1:Digest Insulin 50uL uLmin 60C May 17 Run 1.lcd Detector A:214nm Data2:Digest Insulin 50uL uLmin 60C May 17 Run 3.lcd Detector A:214nm Data3:Digest Insulin 50uL uLmin 60C May 17 Run 2.lcd Detector A:214nm Data5:Digest Insulin 50uL uLmin 60C May 17 Run 3.lcd Detector A:214nm GFFYTPK Undigested Protein minute 2 minutes 4 minutes 8 minutes min Protein Product 14 Sample: Column 1: Column 2: Mobile Phase A: Mobile Phase B: Detection: 5ug insulin Perfinity Optimized Trypsin Column HALO 2.1x100mm RPC 2% ACN, 98% water, 0.1% Formic Acid 90% ACN, 10% water, 0.1% Formic Acid UV/VIS at 214nm The Perfinity Way

15 Automated Digests w/ CV s < 10% uv(x1,000,000) min Peak Number Run Run Run Run Average StDev CV(%)

16 Automated Protein Digests Run 5 Run 10 Run 15 Run 20 16

17 Zero Carryover uv Data1:10uL R&A Tr mm Halo 2pt1 x20 300A GRD no urea R1 Apr 1.lcd PDA Ch1:214nm,4nm(1.00) Data2:blank digestion run.lcd PDA Ch1:214nm,4nm(1.00) min 17

18 What About the Needle in the Haystack?

19 Chromatography Immunoassay An Uncompromised Approach G N M R L H H D M A N Q G N M R L H H D M A N Q K High selectivity (+) Antibodies can be used to isolate proteins from biological extracts. K L L Poor Resolution (-) Immunological contact areas (epitopes) are very small + Poor selectivity (-) Proteins must be extracted or samples fractionated prior to analysis Reverse phase separation of insulin variants, some of which differ by a single amino acid. High resolution (+) Similar proteins differing by small changes in structure can easily be resolved.

20 Perfinity Workstation: Affinity Selection A sample avidin avidin soln. column H + column [B-Ab:Ag] + -[Av 4 ] -[Av 4 :B-Ab:Ag] -[Av 4 :B-Ab] + [Ag] K d = M = 1 fm Universal affinity column RPC trypsin digestion RPC Enhancing Capture Kinetics Allow binding to occur in solution Force antigens through an -Av tunnel Antigen(s) eluting from avidin column. transferrin B -Av -Av -Av Av- Av- Av nm - [Av] times larger than [B-Ab] minute RPC

21 AU % High Volume Column Washing Benefits Affinity sorbent stationary phase = -Protein G:Ab:Ag; 2 column volume wash. Antigen released from Ab with ph 2.5 eluent Without abundant protein removal Note: Before any washing these peaks would have been in x excess of the transferrin peak Transferrin Low affinity proteins are still bound to the protein G column. Immunoglobulin s Minutes 0

22 High Volume Column Washing Benefits Affinity sorbent stationary phase = -Protein G:Ab:Ag; 40 column volume wash Antigen released from Ab with ph 2.5 eluent Transferrin The binding constant of transferrin is so much larger than the non-specifically bound proteins they are washed away at high wash volumes. Desorption of weakly bound proteins can occur with 60 to 120 seconds of washing. HSA

23 Integrated Proteomics Processes Affinity Selection Buffer Exchange and Digest Desalt Peptide Map 3.50 uv(x1,000,000) min 23

24 Perfinity Workstation Pump Waste Trypsin Buffer Exchange Waste Blank Waste Affinity Valve 1 Valve 2 Valve UV/Vis RPC Perfinity Workstation Plumbed for - Affinity Selection Buffer Exchange Trypsin Digestion Desalting Reverse Phase Separation Waste Outlet to MS or Waste Waste Pump Desalt Pump

25 Perfinity Workstation Simple software you don t have to be an LC expert to use the system Step 1: define the method parameters 25 Step 2: create the sample table Step 3: Start the experiment

26 Perfinity Workstation Automates and integrates five key proteomics workflow steps: Affinity Selection, Buffer Exchange, Trypsin Digestion, Desalting & Reverse Phase HPLC. Reduces sample preparation times from 72 hours to < 1hour. Achieves exceptional reproducibility (CVs < 10%) 26

27 Mass Spec Detection (Immuno-MS) Can be coupled with mass spectrometric detection Two different ways of generating ions from the sample: 1. Electrospray ionisation (ESI) online detection 2. MALDI offline Can perform MS to detect all masses e.g. peptide masses» Identify/quantitate proteins present in the sample With the Shimadzu LCMS-8030, can perform targeted acquisitions to detect and quantify known components (increased sensitivity, selectivity) 28

28 Application: Identification of Hb Variants LCMS-IT-TOF - Structural Identification of targets MASCOT Results Rapid identification of VHLTPVEK sickle cell peptide isoform by IT-TOF 91% sequence coverage Rapid identification of sickle cell variants Fully automated extraction, digestion and analysis 29

29 Quantitation: Sickle Cell Anemia Hb LCMS Quantitation of purified targets -Selected ion monitoring 30

30 Benefits of the Perfinity Workstation Three day workflow complete in under 1 hour Serum to purified peptides in < 10 minutes High quality, reproducible (CVs < 10%) Hand free operation fully automated Easily customizable

31 Acknowledgements Perfinity Biosciences Fred Regnier Kevin Meyer Nick Herold Steve Plump THANK YOU! Shimadzu Terry Adams Curtis Campbell Rachel Lieberman Jason Harrington Tom Hayes PerfinityWorkstation.com

32 Summary structural levels of analysis protein level peptide level unit operations A B C D E F G H I J reduction alkylation immune complex formation immune complex capture proteolysis enrich & desalt RPC MS CID MS 30+ min 30+ min 15+ min 1-2 min 1-8 min 1-5 min min <sec <sec <sec 1 st D 2 nd D 3 rd D analytical dimensions 4 th D 5 th D 6 th D Steps Performed in Batch Mode Prior to Injection Steps Performed Sequentially After Injection 34