Presentation on theme: "Practical High Sensitivity LC-MS Fundamentals, Challenges, and Prospects Gary A. Valaskovic, Ph.D. New Objective, Inc."— Presentation transcript:
Practical High Sensitivity LC-MS Fundamentals, Challenges, and Prospects Gary A. Valaskovic, Ph.D. New Objective, Inc.
Main Topics Anatomy of Electrospray Introduction to Nanospray The Nanobore LC Advantage Flow Splitting and Sample Injection Nanobore LC to MS Interfacing Keys to Success
Anatomy of ESI Adapted from Kebarle & Tnag, Anal. Of Chem., 1993, 64, 972A
Anatomy of ESI
What is Nanospray? Flavor of ESIFlow RateSheath Gas Conventional50 to 1000 µL/minYes Microspray0.1 to 10 µL/minOptional Nanospray<0.01 to 0.2 µL/minNot usually
Why Use Nanospray? ESI-MS (as commonly implemented) is a concentration sensitive detector. There is little or no loss in signal/noise as you reduce the flow rate. You can obtain the same S/N for most compounds from 1 mL/min to 10 nL/min (with the right equipment)! Adapted From Cody, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.
Why Use Nanospray? Sensitivity Nanospray is one of the key technologies for MS-based Proteomics There are three reasons to use Nanospray:
How Does Nanospray Yield Sensitivity? Two ways to obtain sensitivity with Nanospray: Off-line Static Nanospray Extend the analysis time for a given sample –Sum spectra to increase S/N –Complete MS/MS or MS n possible On-line LC-Nanospray Analyze a small volume sample (1 µL or much less) –Concentrate your sample into as small a volume as possible
Static Nanospray Methodology Direct infusion of 0.5 to 5 µL sample Sample must be clean No pumps - flow is generated by electrostatic pressure Typical Tip ID: 1 - 4µm Typical flow rate: nL/min MS Inlet Tip ID µm Glass needle mm bore Conductive Coating Liquid sample µL HV
Static Nanospray Extends Analysis Time Adapted From Corey & Pinto, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.
Static Nanospray Limitations Sensitivity is good, but inferior to LC methods –Typically fmol proteins and peptides Sample prep is not integral, sample must be clean and concentrated –Typically 100 nM to 10 µM Limited utility on complex mixtures (OK on single bands but unable to handle shotgun methods) Highly dependent on operator skill Limited throughput Automation is possible but $$$
On-line Nanospray with Nanobore LC Integral sample clean-up On-line injection of µL Gradient elution from split flow HPLC pump Column ID 100 µm Typical flow rate: nL/min Column MS Inlet In-line filter Flow split 1000:1 Micro-injection valve (or autosampler) Gradient 200 µL/min Tip
Why Use Nanospray LC? 4.6 mm 50 µm Elute your sample into the smallest practical volume for the highest S/N!
Requirements for LC System Gradient Operation Binary required; tertiary, quaternary preferred Injection µL Typical Accommodate sample trapping Flow rate 100 to 1000 nL/min Typically pre-column flow split from conventional pump
Flow Splitting Methods Simple T Splitter (build) Inexpensive! Easy to do. Split is non-linear but reproducible. Balanced Flow Splitter (build or buy) Good performance, inexpensive High-Pressure Flow Splitter (buy) Good performance, $$$ Active Mass Flow Control (buy) Good performance, $$$
Simple Flow Splitting Use a simple Tee Use a small bore ( µm ID) tubing to create a flow calibrator Adjust split ratio by adjusting the length of the calibrator Fine tune by setting the pump flow Ratios from 1:10 to 1:1000 are readily obtained
Nanospray Source Requirements Mechanical requirements –XYZ Stage for tip positioning –Tip and spray imaging system –Junction and proximal HV contact Tip requirements –ID of µm –Typically fused-silica, 360 µm OD –Uncoated or coated
On-line Nanospray Source Objective Lens CCD Camera Injection Valve Tip Holder HV Contact XYZ Stage m
What About Sample Injection? Gradient elution in reverse phase enables sample stacking: Large ( µL) injection volumes are OK If we ran isocratically, a 75 µm ID column would require a nL injection volume!
Injection Strategies On-column Injection (Pressure Bomb) –High sensitivity –Zero sample loss or waste –Time consuming (manual) Micro Injection Valve – µL –Easy to use Sample Trapping –Faster injection of large volumes ( µL) –Trap protects columns for increased lifetime –Some peptides lost during injection and analysis
Bomb Injection Pressure Bomb To Column Gas In Sample Vial
Sample Trapping Trap Cartridge/Column – µm ID – mm in length Typically C18 or SCX Loading rate µL/min Enable hundreds/thousands of injections on an analytical column Fused Silica Column
Sample Trapping Load Injection Loop
Sample Trapping Load Sample Trap & Wash
Sample Trapping Elute into Column
How Do We Interface? Liquid sheath for make-up flow (The Early Days) –Generally not used, compromised sensitivity Direct Connect interface with fused-silica tip –No make-up or sheath liquid –Reasonable sensitivity –Plumbing can be a challenge Integration of LC column with emitter –Highest sensitivity –Robust interface –Greater ease of use
Direct Connect Interface Junction Contact ZDV Metal Union Union PEEK or Teflon Distal Coating HV Tip µm HV
Performance Benchmark Tryptic Digest of BSA fmol Base Peak, RIC SIC, m/z SIC, µm ID, C18 Distal Coated 10 µm PicoTip Water/CH3CN/Formic Acid 45 Minute gradient Micromass Q-TOF Data courtesy Art Moseley, GlaxoSmithKline
Direct Connect Interface Common Problems Poor peak shape Difficult post-column plumbing, requiring a perfect connection Impractical with columns smaller than 75 µm Clogged tips and columns Difficult to distinguish point of plug - is it the column or the tip? Air bubbles in line Out-gassing, leaks, electrolysis, etc.
PicoFrit Packed Tip Performance Emmett & Caprioli, J. Am. Soc. Mass. Spec. 1994, 5, Pack the LC column directly into the tip! Zero post column volume 75 µm ID, C18FritTip: µm
PicoFrit Packed Tip Approach Junction style HV contact for robustness (arc immunity) Junction can be far behind tip (10 cm or more) Pre-column volume does not hurt chromatography Pt electrode PEEK T HV Packed C18
PicoFrit Approach Analytical Advantages Tip size optimal for column flow rate –Typically µm for 75 µm ID column HV contact on inlet side of column –Minimal contribution to band broadening w/sample stacking –Eliminates air bubbles (high pressure side of column) –Robust and easy to use Economical –Concurrent fabrication of tip and column
Packed Tip Appraoch Analytical Advantages Optimal sensitivity and resolution –Spray directly from column –Virtually zero post-column volume Virtually eliminates tip clogging –Robust lifetime –500+ injections/column with sample trapping Easy to use –Fewer connections to make
PicoFrit Columns Performance Benchmark Data courtesy James P. Murphy III, Ph.D.
PicoFrit Columns Performance Benchmark
Keys to Success
Minimize Particle Contamination
Mobile Phase Stocks Change Stocks Regularly (weekly or better) Use bottled water, preferrably distilled in glass Avoid ultrpure meg-ohm water from in-house systems –These can contain high levels of carbon particulates Contaminated Column HeadClean Column Head Poor quality water is the primary cause of clogged columns!
Minimize Particle Contamination Fittings and Unions Use PEEK or FEP adapter sleeves Dont over tighten fittings Avoid graphitized ferrules (common in GC) Discard contaminated fittings OUCH!
Minimize Particle Contamination Injection valves Avoid scribing surface of rotor with fused-silica Inspect surfaces often Pump components Inspect/replace seals, fittings, check valves and filters Watch out!
Measuring Column Flow Rate Let a droplet collect at tip for 5-10 minutes (ESI is off) Collect the droplet by capillary action Measure the volume and calculate flow rate
Source Tuning: Go For the Best Spray 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit tip, LCQ Deca XP Inlet 850V Stream and Plume
Source Tuning: Go For the Best Spray 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit tip, LCQ Deca XP Inlet 850V Stream and Plume 1150V Stream and Plume
Source Tuning: Go For the Best Spray 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit tip, LCQ Deca XP Inlet 850V Stream and Plume 1450V Good Plume
Source Tuning: Go For the Best Spray 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit tip, LCQ Deca XP Inlet 850V Stream and Plume 1850V Optimal Plume
Source Tuning: Go For the Best Spray 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit tip, LCQ Deca XP Inlet 850V Stream and Plume 2050V Split Plume
Source Tuning: Challenges Spray characteristics are sensitive to: Emitter size, shape, distance Flow rate Voltage Mobile phase composition –Optimal results require a changing voltage! Bottom line: Tune your spray under eluting conditions
Performance Benchmarks Cell mapping project at McGill University Daniel Boismenu, Montréal Network for Pharmaco-Proteomics and Structural Genomics Exhaustive proteomic analysis of cell organelles Determine elation between protein function and location Total of D lanes for cell map: 93 slices per lane Total of 125,550 slices 1 hour of HPLC-MS/MS per gel slice 5231 days of instrument time = 14 years
Performance Benchmarks Robustness Data courtesy Daniel Boismenu, McGill University Injection #31: Plasma membrane challenged with insulin. In gel digestion of slice no 30 of µm x 10 cm C18 PicoFrit column, with 300 µm x 1 mm C18 Trap Cartridge on Micromass Q-TOF
Performance Benchmarks Robustness Data courtesy Daniel Boismenu, McGill University Injection #881: Smooth endoplasmic reticulum, aqueous phase. In gel digestion of slice no 45 of 92 (Over 1 month of continuous, 24 hr, 7 days/week operation) … and still going!
Keys to Success with Nanobore LC-MS Clean mobile phase –Minimize particulate contamination –Use multiple high quality in-line filters Know your flow rate –Monitor through column flow periodically Use the right injection scheme for your samples Throughput vs. sensitivity Minimize (or eliminate) post-column plumbing –Use special care with post-column connections –Use a tip-column (PicoFrit) format Optimize electrospray conditions –Stabilize spray with voltage –Maximize S/N with emitter position –Match tip size to flow rate