practical high sensitivity lc-ms fundamentals, challenges, and prospects gary a. valaskovic, ph.d....
TRANSCRIPT
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Practical High Sensitivity LC-MS
Fundamentals, Challenges, and Prospects
Gary A. Valaskovic, Ph.D.
New Objective, Inc.
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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
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Anatomy of ESI
Adapted from Kebarle & Tnag, Anal. Of Chem., 1993, 64, 972A
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Anatomy of ESI
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What is Nanospray?
Flavor of ESI Flow Rate Sheath Gas
Conventional 50 to 1000 µL/min Yes
Microspray 0.1 to 10 µL/min Optional
Nanospray <0.01 to 0.2 µL/min Not usually
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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.
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Why Use Nanospray?
Sensitivity
Sensitivity
Sensitivity
Nanospray is one of the key technologies for MS-based Proteomics
There are three reasons to use Nanospray:
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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 MSn 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
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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: 10 - 50 nL/min
MS Inlet
Tip ID 1 - 4 µm
Glass needle - 0.7 mm bore Conductive Coating
Liquid sample1 - 5 µL
HV
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Static Nanospray Extends Analysis Time
Adapted From Corey & Pinto, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.
Conventional ESI Flow Injection1µL Sample Injection@ 10 µL/min
Nanospray1 µL Sample≈ 30 nL/min
Time (min)
6 S FWHM
10 1550
100%
20 25 30 35 40
10 1550
100%
20 25 30 35 40
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Static Nanospray Limitations
• Sensitivity is good, but inferior to LC methods– Typically 10 -100 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 $$$
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“On-line” Nanospray with Nanobore LC
• Integral sample clean-up
• On-line injection of 1 - 20 µL
• Gradient elution from split flow HPLC pump
• Column ID ≤ 100 µm
• Typical flow rate: 100 - 500 nL/min
Column
MS Inlet
In-line filter
Flow split1000:1
Micro-injection valve(or autosampler)
Gradient pump@ 200 µL/min
Tip
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Why Use Nanospray LC?
4.6 mm
50 µm
Elute your sample into the smallest practical volume for the highest S/N!
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Why Use Nanobore LC?
Column ID Flow Rate Relative [C]
Standard 4.6 mm 1 mL/min 1
Microbore 1 mm 50 µL/min 21
Capillary 320 µm 5 µL/min 206
Nanobore 75 µm 250 nL/min 3,750
Nanobore 50 µm 150 nL/min 8,450
The
Concentration Advantage!
Adapted From Tomer & Moseley, Mass. Spec. Rev., 1994, 13, 431
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Requirements for LC System
Gradient Operation• Binary required; tertiary, quaternary preferred
Injection• 1 - 20 µL Typical
• Accommodate sample trapping
Flow rate ≈ 100 to 1000 nL/min• Typically pre-column flow split from conventional pump
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Flow Splitting Methods
Simple “T” Splitter (build)• Inexpensive! Easy to do. Split is non-linear but
reproducible.
Balanced Flow Splitter (build or buy)• Good performance, inexpensive
High-Pressure Flow Splitter (buy)• Good performance, $$$
“Active” Mass Flow Control (buy)• Good performance, $$$
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Simple Flow Splitting
• Use a simple Tee
• Use a small bore (20 - 50 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
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Nanospray Source Requirements
• Mechanical requirements– XYZ Stage for tip positioning– Tip and spray imaging system– Junction and proximal HV contact
• Tip requirements– ID of 10 - 30 µm– Typically fused-silica, 360 µm OD– Uncoated or coated
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On-line Nanospray Source
Objective Lens
CCD Camera
Injection Valve
Tip Holder
HV Contact
XYZ Stage
www.newobjective.com
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On-line Nanospray Source
Monitor
Illuminator
Source
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What About Sample Injection?
Gradient elution in reverse phase enables sample stacking:
• Large (1 - 20 µL) injection volumes are OK
If we ran isocratically, a 75 µm ID column would require a 10 - 20 nL injection volume!
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Injection Strategies
• On-column Injection (Pressure Bomb)– High sensitivity– Zero sample loss or waste– Time consuming (manual)
• “Micro” Injection Valve– 0.1 - 5 µL– Easy to use
• Sample Trapping– Faster injection of large volumes (5 - 20 µL)– Trap protects columns for increased lifetime– Some peptides lost during injection and analysis
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Bomb Injection
Pressure Bomb
To Column
Gas In
Sample Vial
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Sample Trapping
• Trap Cartridge/Column– 100 - 500 µm ID
– 1 - 25 mm in length
• Typically C18 or SCX
• Loading rate 1 - 20 µL/min
• Enable hundreds/thousands of
injections on an analytical
column
Fused Silica Column
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Sample Trapping
Load Injection Loop
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Sample Trapping
Load Sample Trap & Wash
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Sample Trapping
Elute into Column
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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
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Direct Connect InterfaceJunction Contact
ZDV Metal Union
UnionPEEK or Teflon
Distal Coating
HV Tip5 - 30 µm
HV
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Performance BenchmarkTryptic Digest of BSA - 125 fmol
Base Peak, RIC
SIC, 653.5 m/z
SIC, 653.5 75 µm ID, C18
Distal Coated 10 µm PicoTip™
Water/CH3CN/Formic Acid
45 Minute gradient
Micromass Q-TOF
Data courtesy Art Moseley, GlaxoSmithKline
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Direct Connect InterfaceCommon 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.
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PicoFrit™ Packed Tip Performance
Emmett & Caprioli, J. Am. Soc. Mass. Spec. 1994, 5, 605-613
Pack the LC column directly into the tip!
“Zero” post column volume
75 µm ID, C18 Frit Tip: 8 - 15 µm
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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
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PicoFrit™ ApproachAnalytical Advantages
• Tip size optimal for column flow rate– Typically 8 -15 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
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Packed Tip AppraochAnalytical 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
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PicoFrit™ ColumnsPerformance Benchmark
Data courtesy James P. Murphy III, Ph.D.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36Time (min)
0
20
40
60
80
100
5 -10 fmol/peptide Angiotensin mixture1µL Bomb Injection
RIC full scan 300 - 1500 m/zProteoPep C18 75µm ID PicoFrit column
Impurity
*
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PicoFrit™ ColumnsPerformance Benchmark
1000 1100 1200 1300 1400 1500800 900m/z
300 400 500 600 700
521.0 565.2
332.0460.5349.0
0
459.4
441.1
564.2
(M + 2H)2+
604.2 648.1
773.8693.2
917.4
918.5
919.2
(M + H)+
861.1793.7
NL: 2.57 E7
Full Scan MS
Peak #3
RT: 25.84 - 26.29
1160.01134.41035.5 1179.0 1255.4
1402.4
1306.11499.51424.31384.9
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Keys to Success
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Minimize Particle Contamination
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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 Head Clean Column Head
Poor quality water is the primary cause of clogged columns!
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Minimize Particle Contamination
Fittings and Unions• Use PEEK or FEP adapter sleeves• Don’t over tighten fittings• Avoid graphitized ferrules (common in GC)• Discard contaminated fittings
OUCH!
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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!
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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
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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
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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 Plume1150V Stream and Plume
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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 Plume1450V Good Plume
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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 Plume1850V Optimal Plume
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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 Plume2050V Split Plume
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Spray Morphology: Composition
5% ACN 50% ACN 95% ACN
1700V
1900V
2100V
2300V
2500V
3100V30 µm Tip @ 500 nL/min
0.1% Formic Acid
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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”
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Performance Benchmarks
Cell mapping project at McGill UniversityDaniel Boismenu, Montréal Network for Pharmaco-Proteomics and Structural Genomics
Exhaustive proteomic analysis of cell organelles
Determine elation between protein function and location
Total of 1350 1-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
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Performance BenchmarksRobustness
Data courtesy Daniel Boismenu, McGill University
Injection #31: Plasma membrane challenged with insulin.In gel digestion of slice no 30 of 6475 µm x 10 cm C18 PicoFrit™ column, with 300 µm x 1 mm C18 Trap Cartridge on Micromass Q-TOF
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Performance BenchmarksRobustness
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!
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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
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