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