Analyzing Biological and Analyzing Biological and Organic Polymers by Organic Polymers by

MALDI-TOFMALDI-TOFJonathan A. Karty, Ph.D.Jonathan A. Karty, Ph.D.

Topics CoveredTopics CoveredSample RequirementsSample Requirements

Instrument OverviewInstrument Overview

General Instrument Use InstructionsGeneral Instrument Use Instructions

Tips and TricksTips and Tricks

What is the Bruker Autoflex III?What is the Bruker Autoflex III? Time-of-flight mass spectrometerTime-of-flight mass spectrometer

Ions of given same kinetic energy, heavy ions travel Ions of given same kinetic energy, heavy ions travel slower than lighter onesslower than lighter ones

Two modes of operationTwo modes of operation LinearLinear ReflectronReflectron

MALDI/LDI sourceMALDI/LDI source 384 position target plate (~1 384 position target plate (~1 µµL spot size)L spot size) 355 nm Nd:YAG laser355 nm Nd:YAG laser Can analyze positive or negative ions (same spot)Can analyze positive or negative ions (same spot)

Autoflex III PictureAutoflex III Picture

Matrix-Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption/Ionization (MALDI)(MALDI)

Analyte is mixed with UV-absorbing matrixAnalyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio~10,000:1 matrix:analyte ratio Analyte does not need to absorb laserAnalyte does not need to absorb laser

A drop of this liquid is dried on a targetA drop of this liquid is dried on a target Analyte incorporated into matrix crystalsAnalyte incorporated into matrix crystals

Spot is irradiated by a laser pulseSpot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with itIrradiated region sublimes, taking analyte with it Matrix is often promoted to the excited stateMatrix is often promoted to the excited state Charges exchange between matrix and analyte in the Charges exchange between matrix and analyte in the

plume (very fast <100 nsec)plume (very fast <100 nsec) Ions are accelerated toward the detectorIons are accelerated toward the detector

MALDI DiagramMALDI Diagram

Image from http://www.noble.org/Plantbio/MS/iontech.maldi.html

MALDI AdvantagesMALDI Advantages Technique is relatively simpleTechnique is relatively simple Volatilize and ionize labile molecules Volatilize and ionize labile molecules

Imagine electron ionization on a proteinImagine electron ionization on a protein MALDI creates very simple mass spectraMALDI creates very simple mass spectra

Ions are usually (M+nH)Ions are usually (M+nH)n+n+ or (M-nH) or (M-nH)n-n-

Only 1-3 charge states are observedOnly 1-3 charge states are observed Usually 1 charge state for peptides < 3.5 kDaUsually 1 charge state for peptides < 3.5 kDa

MALDI ideal for time-of-flight analyzersMALDI ideal for time-of-flight analyzers Theoretically unlimited mass range (100 kDa done here)Theoretically unlimited mass range (100 kDa done here)

MALDI is very rapid (<1 min/spot)MALDI is very rapid (<1 min/spot) Low sample consumption (1 Low sample consumption (1 µµL)L) Wide array of matrices available for different analytesWide array of matrices available for different analytes

Some Common MALDI MatricesSome Common MALDI MatricesC

N

CO

OH

HO O

O

HO

H3C

CH3

CO

OH

C

O

OH

OH

HO

NH

C

O

OHC

N

C

N

C

N

C

N

-cyano-4-hydroxycinnamic acidCCA C10H7NO3

peptides and small molecules

sinapinic acidSA C11H12O5

proteins

2,5 dihydroxybenzoic acidDHB C7H6O4

oligosaccharides

N

3-indoleacrylic acidIAA C10H9NO2

organic polymers

7,7,8,8 tetracyanoquinodimethaneTCNQ C12H4N4

polyaromatic hydrocarbons

CO

OH

OH

3-hydroxypicolinic acidHPA C6H5NO3oligonucleotides

What Samples Can It Run?What Samples Can It Run? BiopolymersBiopolymers

Peptides, proteins, DNA, RNA, oligosaccharidesPeptides, proteins, DNA, RNA, oligosaccharides Organometallic complexesOrganometallic complexes

Organometallic salts work greatOrganometallic salts work great Synthetic polymersSynthetic polymers

Polymer need not be soluble in same solvent as Polymer need not be soluble in same solvent as matrixmatrix

Molecules that photoionize upon irradiation by Molecules that photoionize upon irradiation by 355 nm laser355 nm laser PorphyrinsPorphyrins Organometallic complexesOrganometallic complexes

What Samples Can’t It Run?What Samples Can’t It Run? ““Dirty” samplesDirty” samples

Significant concentration of involatilesSignificant concentration of involatiles Glycerol, urea, most buffers, many detergentsGlycerol, urea, most buffers, many detergents

Alkali metal salts can be quite problematicAlkali metal salts can be quite problematic RNA/DNA analyses require extensive desaltingRNA/DNA analyses require extensive desalting

Molecules with significant vapor pressuresMolecules with significant vapor pressures Instrument is held at ~10Instrument is held at ~10-7-7 torr torr

Molecules that do not make stable ions in Molecules that do not make stable ions in sourcesource Lack charge acceptor/donor siteLack charge acceptor/donor site Cannot photoionize with Nd:YAG laserCannot photoionize with Nd:YAG laser

MALDI AdvantagesMALDI AdvantagesRelatively gentle ionization techniqueRelatively gentle ionization techniqueVery high MW species can be ionizedVery high MW species can be ionizedMolecule need not be volatileMolecule need not be volatileVery easy to get sub-picomole sensitivityVery easy to get sub-picomole sensitivitySpectra are easy to interpretSpectra are easy to interpretPositive or negative ions from same spotPositive or negative ions from same spotWide array of matrices availableWide array of matrices available

MALDI DisadvantagesMALDI DisadvantagesMALDI matrix cluster ions can obscure low MALDI matrix cluster ions can obscure low

m/z (<600) rangem/z (<600) rangeAnalyte must have very low vapor pressureAnalyte must have very low vapor pressureCoupling MALDI with chromatography can Coupling MALDI with chromatography can

be difficultbe difficultAnalytes that absorb the laser can be Analytes that absorb the laser can be

problematicproblematicFluorescein-labeled peptidesFluorescein-labeled peptides

Instrument DiagramInstrument Diagram

ReflectorDetector

LinearDetectorLens

Target

ExtractionPlate

FlightTube

Entrance

Reflectron

355 nm Nd:YAG laser

Linear ModeLinear Mode

ReflectorDetector

LinearDetectorLens

Target

ExtractionPlate

FlightTube

Entrance

Reflectron

355 nm Nd:YAG laser

Linear mode is used for large (>3.5 kDa) molecules or

exceedingly fragile species (oligosaccharides). It is capable of 4,000 resolving power @ 3.2

kDa (1,000 RP @ 12 kDa)

Reflectron ModeReflectron Mode

ReflectorDetector

LinearDetectorLens

Target

ExtractionPlate

FlightTube

Entrance

Reflectron

355 nm Nd:YAG laser

Reflectron mode is used for small species (<4 kDa) and is capable of 11,000 resolving

power @ 3.2 kDa.

MALDI ExampleMALDI Example

(Ubiq+H)+(Ins+H)+(U

biq+

2H)2+

(ACTH 7-38+H)+(A

CTH

18-

37+H

)+

O

N

N

O

HN

HN

O

O

C12H25O

C12H25O

C12H25O

C12H25O

OC12H25

OC12H25

O O OO

N

N

O

NH

NH

O

O

OC12H25

OC12H25

OC12H25

OC12H25

C12H25O

C12H25O

Chemical Formula: C230H354N8O23Exact Mass: 3596.68

MALDI Example I ContinuedMALDI Example I Continued

8476.61

16952.53

4802.98

0

2000

4000

6000

Inten

s. [a.

u.]

5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 m/z

16952.53

17131.5417056.51

0

1000

2000

3000

4000

Inten

s. [a.

u.]

16600 16800 17000 17200 17400 17600 m/z

MALDI-TOF Example 2MALDI-TOF Example 2

General Sample GuidelinesGeneral Sample Guidelines Purify analyte if possiblePurify analyte if possible

Analyte should be 5 – 100 Analyte should be 5 – 100 µµM in concentrationM in concentration ZipTips can help purify dirty samples (CZipTips can help purify dirty samples (C44 and C and C1818

available in MSF)available in MSF) Use only volatile solvents/buffersUse only volatile solvents/buffers

MeOH, HMeOH, H22O, acetone, CHO, acetone, CH33CN, THF, CHCN, THF, CH22ClCl22, C, C66HH66

TFA, HOAc, formic acid, NHTFA, HOAc, formic acid, NH33, etc., etc. Ionic strength < 20 mM (e.g. 0.1% v/v HOAc)Ionic strength < 20 mM (e.g. 0.1% v/v HOAc)

If you need a detergent, 20 mM n-If you need a detergent, 20 mM n-octylglucopyranoside can workoctylglucopyranoside can work No SDS, TWEEN, CHAPS, etcNo SDS, TWEEN, CHAPS, etc

Need at least 2 Need at least 2 µµLL

Sample Prep TricksSample Prep Tricks Ziptip to clean up dirty samplesZiptip to clean up dirty samples

CC1818 for peptides < 3 kDa for peptides < 3 kDa CC44 for peptides/proteins > 3kDa for peptides/proteins > 3kDa Elute directly into matrix for added sensitivityElute directly into matrix for added sensitivity ZipTip instructions on MSF websiteZipTip instructions on MSF website

If CCA liquid turns yellow, pH is too highIf CCA liquid turns yellow, pH is too high Spots from non-acidic CCA do not crystallize correctlySpots from non-acidic CCA do not crystallize correctly Add a little 1% v/v or 10% v/v TFA to lower pHAdd a little 1% v/v or 10% v/v TFA to lower pH If sample needs base for solubility, try over-layer If sample needs base for solubility, try over-layer

methodmethod Dissolve sample in NHDissolve sample in NH33 or other volatile base or other volatile base Place 1 uL of sample on target, let dry completelyPlace 1 uL of sample on target, let dry completely Deposit 1 uL matrix over top of dried sampleDeposit 1 uL matrix over top of dried sample

Sample Prep Tricks 2Sample Prep Tricks 2Non-aqueous over-layerNon-aqueous over-layer

Make 1 uL spot of matrix on plate, let dryMake 1 uL spot of matrix on plate, let dryDeposit small amount of sample in volatile Deposit small amount of sample in volatile

solvent (e.g. CHClsolvent (e.g. CHCl33, acetone, CH, acetone, CH22ClCl22))You can even do internal calibration this wayYou can even do internal calibration this way

Put calibrants in matrix spotPut calibrants in matrix spotFor better mass accuracy, let voltages For better mass accuracy, let voltages

stabilize 10-30 minutes before recording stabilize 10-30 minutes before recording datadata

Hands-on TrainingHands-on TrainingStarts AFTER 11/7Starts AFTER 11/7Groups of no more than threeGroups of no more than threeOne hour or so to completeOne hour or so to completeNo charge for first sessionNo charge for first sessionAfter training, students must demonstrate After training, students must demonstrate

competency by running their own samples competency by running their own samples prior to being granted after-hours accessprior to being granted after-hours access