Transcript Practical, High Sensitivity LC-MS

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?
There are three reasons to use Nanospray:
Sensitivity
Sensitivity
Sensitivity
Nanospray is one of the key technologies
for MS-based Proteomics
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
HV
MS Inlet
Glass needle - 0.7 mm bore
Liquid sample
1 - 5 µL
Conductive
Coating
Tip ID 1 - 4 µm
Static Nanospray Extends Analysis Time
100%
Conventional ESI Flow Injection
1µL Sample Injection
@ 10 µL/min
6 S FWHM
0
5
10
15
20
25
30
35
40
100%
Nanospray
1 µL Sample
Å 30 nL/min
0
5
10
15
20
25
Time (min)
30
35
40
Adapted From Corey & Pinto, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.
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 $$$
“On-line” Nanospray with Nanobore LC
• Integral sample clean-up
• On-line injection of 1 - 20 µL
• Gradient elution from split flow HPLC pump
• Column ID ≤ 100 µm
• Typical flow rate: 100 - 500 nL/min
Gradient pump
@ 200 µL/min
MS Inlet
In-line filter
Column
Tip
Flow split
1000:1
Micro-injection valve
(or autosampler)
Why Use Nanospray LC?
4.6 mm
50 µm
Elute your sample into the smallest practical volume for the highest S/N!
Why Use Nanobore LC?
The
Concentration Advantage!
Column
ID
Flow Rate
Relative [C]
Standard
4.6 mm
1 mL/min
1
Microbore
1 mm
50 µL/min
21
Capillary
320 µm
5 µL/min
206
Nanobore
75 µm
250 nL/min
3,750
Nanobore
50 µm
150 nL/min
8,450
Adapted From Tomer & Moseley, Mass. Spec. Rev., 1994, 13, 431
Requirements for LC System
Gradient Operation
• Binary required; tertiary, quaternary preferred
Injection
• 1 - 20 µL Typical
• Accommodate sample trapping
Flow rate ≈ 100 to 1000 nL/min
• Typically pre-column flow split from conventional pump
Flow Splitting Methods
Simple “T” Splitter (build)
• Inexpensive! Easy to do. Split is non-linear but
reproducible.
Balanced Flow Splitter (build or buy)
• Good performance, inexpensive
High-Pressure Flow Splitter (buy)
• Good performance, $$$
“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
Tip Holder
HV Contact
CCD Camera
XYZ Stage
Injection Valve
www.newobjective.co
m
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
To Column
Pressure Bomb
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 Interface
Junction Contact
HV
ZDV Metal Union
HV
Union
Distal Coating
PEEK or Teflon
Tip
5 - 30 µm
Performance Benchmark
Tryptic Digest of BSA - 125 fmol
SIC, 653.5 m/z
Base Peak, RIC
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 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
75 µm ID, C18
Frit
Tip: 8 - 15 µm
Pack the LC column directly into the tip!
“Zero” post column volume
Emmett & Caprioli, J. Am. Soc. Mass. Spec. 1994, 5, 605-613
PicoFrit™ Packed Tip Approach
HV
Pt electrode
PEEK “T”
Packed C18
• 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
PicoFrit™ Approach
Analytical 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 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
5 -10 fmol/peptide
Angiot ensin mixture
1µL Bomb Inj ection
*
RIC full scan 300 - 1500 m/z
ProteoPep C18 75µm ID PicoFrit column
100
80
60
Impuri ty
40
20
0
0
2
4
6
8
10
12
14
16
18
20
T ime (min)
22
24
26
28
30
32
34
36
Data courtesy James P. Murphy III, Ph.D.
PicoFrit™ Columns
Performance Benchmark
(M + 2H) 2+
NL: 2.57 E7
459.4
Full Scan MS
Peak #3
441.1
RT: 25.84 - 26.29
564.2
521.0
332.0
565.2
(M + H) +
917.4
349.0
604.2
918.5
648.1
793.7
693.2
0
300
400
1402.4
460.5
500
600
700
861.1
919.2
773.8
800
900
m/z
1035.5
1000
1134.4
1100
1160.0
1179.0
1200
1255.4
1300
1306.1
1384.9
1424.3
1400
1499.5
1500
Keys to Success
Minimize Particle Contamination
Minimize Particle Contamination
Contaminated Column Head
Clean Column Head
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
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
850V Stream and Plume
50% ACN, 0.1% Formic Acid
500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet
Source Tuning: Go For the Best Spray
1150V
Streamand
andPlume
Plume
850V Stream
50% ACN, 0.1% Formic Acid
500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet
Source Tuning: Go For the Best Spray
1450V Good Plume
850V Stream and Plume
50% ACN, 0.1% Formic Acid
500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet
Source Tuning: Go For the Best Spray
1850V Optimal Plume
850V Stream and Plume
50% ACN, 0.1% Formic Acid
500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet
Source Tuning: Go For the Best Spray
2050V Split Plume
850V Stream and Plume
50% ACN, 0.1% Formic Acid
500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet
Spray Morphology: Composition
5% ACN
50% ACN
95% ACN
1700V
1900V
2100V
2300V
2500V
3100V
30 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 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 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 Benchmarks
Robustness
Injection #31: Plasma membrane challenged with insulin.
In gel digestion of slice no 30 of 64
75 µm x 10 cm C18 PicoFrit™ column, with 300 µm x 1 mm C18 Trap Cartridge on Micromass Q-TOF
Data courtesy Daniel Boismenu, McGill University
Performance Benchmarks
Robustness
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!
Data courtesy Daniel Boismenu, McGill University
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