Practical High Sensitivity LC-MS Fundamentals, Challenges, and Prospects Gary A. Valaskovic, Ph.D....
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Transcript of Practical High Sensitivity LC-MS Fundamentals, Challenges, and Prospects Gary A. Valaskovic, Ph.D....
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 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
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
Sensitivity
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 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
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
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
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 $$$
“On-line” Nanospray with Nanobore LC
• Integral sample clean-up
• On-line injection of 1 - 20 µL
• Gradient elution from split flow HPLC pump
• Column ID ≤ 100 µm
• Typical flow rate: 100 - 500 nL/min
Column
MS Inlet
In-line filter
Flow split1000:1
Micro-injection valve(or autosampler)
Gradient pump@ 200 µL/min
Tip
Why Use Nanospray LC?
4.6 mm
50 µm
Elute your sample into the smallest practical volume for the highest S/N!
Why Use Nanobore LC?
Column ID Flow Rate Relative [C]
Standard 4.6 mm 1 mL/min 1
Microbore 1 mm 50 µL/min 21
Capillary 320 µm 5 µL/min 206
Nanobore 75 µm 250 nL/min 3,750
Nanobore 50 µm 150 nL/min 8,450
The
Concentration Advantage!
Adapted From Tomer & Moseley, Mass. Spec. Rev., 1994, 13, 431
Requirements for LC System
Gradient Operation• Binary required; tertiary, quaternary preferred
Injection• 1 - 20 µL Typical
• Accommodate sample trapping
Flow rate ≈ 100 to 1000 nL/min• Typically pre-column flow split from conventional pump
Flow Splitting Methods
Simple “T” Splitter (build)• Inexpensive! Easy to do. Split is non-linear but
reproducible.
Balanced Flow Splitter (build or buy)• Good performance, inexpensive
High-Pressure Flow Splitter (buy)• Good performance, $$$
“Active” Mass Flow Control (buy)• Good performance, $$$
Simple Flow Splitting
• Use a simple Tee
• Use a small bore (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
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
On-line Nanospray Source
Objective Lens
CCD Camera
Injection Valve
Tip Holder
HV Contact
XYZ Stage
www.newobjective.com
On-line Nanospray Source
Monitor
Illuminator
Source
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!
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
Bomb Injection
Pressure Bomb
To Column
Gas In
Sample Vial
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
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 InterfaceJunction Contact
ZDV Metal Union
UnionPEEK or Teflon
Distal Coating
HV Tip5 - 30 µm
HV
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
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.
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
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™ 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
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
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
*
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
Keys to Success
Minimize Particle Contamination
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!
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!
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 Plume1150V 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 Plume1450V 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 Plume1850V 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 Plume2050V Split Plume
Spray Morphology: Composition
5% ACN 50% ACN 95% ACN
1700V
1900V
2100V
2300V
2500V
3100V30 µm Tip @ 500 nL/min
0.1% Formic Acid
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 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
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
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!
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