Sample Preparation

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Sample Preparation
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Basic Sample Preparation

• Matrix selection for different sample classes.
• Methods for matrix preparation.
• Sample preparation methods for different sample
  classes.
• Sample plate types.

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Ideal Sample Concentration for MALDI
Compound
Concentration
Peptides and proteins
0.1 to 10 pmol/µl
Some proteins, particularly glycoproteins, yield
better results at concentrations up to 10 pmol/µl
Oligonucleotides
10 to 100 pmol/µl
Polymers
100 pmol/µl
Note 1 pmol/ul 10-6 M
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Sample Preparation
Select matrix
Prepare matrix
Prepare sample
Mix sample and matrix
Apply to a clean sample plate
Dry
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Matrix selection
a-Cyano-4-hydroxy-cinnamic acid (CHCA)
Peptideslt10kDa
Sinapinic Acid
Proteins gt10kDa
Neutral Carbohydrates,
2,5-Dihydroxybenzoic acid (DHB)
Synthetic Polymers
Super DHB
Proteins,
Glycosylated proteins
3-Hydroxypicolinic acid
Oligonucleotides
Proteins,
2-(4-hydroxy-phenylazo) benzoic acid (HABA)
Oligosaccharides
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• Matrix selection
• a-cyano-4-hydroxycinnamic acid (a-Cyano, CHCA)
• Analytes peptides, protein digests, small
  proteins and
• PNAs below 10,000 Da
• Preparation 10 mg/ml in 50 ACN with 0.1 TFA
  10 mg/ml in 111 H2OACNEtOH. (Note use 0.1
  TFA, keep pH lt 2.)
• Recrystallization may significantly improve
  performance.
• Dissolve CHCA in warm EtOH to saturation.
• Filter, add two volumes of cold water, then let
  stand at 4?C for 24 hr or more in explosion-proof
  fridge
• Filter, wash the precipitate with cold water and
  air dry or dry in lyophilizer

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Matrix selection
Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic
acid)
Analytes proteins, large peptides Preparation
10 mg/ml in 30 ACN with 0.1 TFA or saturated
solution of SA in the same solvent. ACN conc.
can be increased up to 50-70 depending on sample
Note Try the dried droplet method first can
also be used with the crushed crystal method of
sample preparation.
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Appearance of Matrix Crystals
CHCA Rounded
Sinapinic acid Rhomboid
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Matrix selection
DHB (2,5-dihydroxybenzoic acid, gentisic acid)
Analytes small molecules, phosphopeptides,
peptides, neutral carbohydrates, some synthetic
polymers Preparation 10 mg/ml in water, 50 ACN
or other appropriate solvent for analyte
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Results from Different Drying Techniques with DHB

• Dried under vacuum
• Promotes salt adducts
• Homogenous crystallization, easier for automation
• Good for neutral carbohydrates and polymers
• Similar effects to using high organic content in
  the matrix solvent.

• Air dried
• Fewer salt adducts
• Heterogeneous crystallization, spot-to-spot
  variations
• Good for peptides and proteins

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DHB Crystallization
Vacuum dried Milky, amorphous
Air dried Irregular crystals in ring
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Synthetic Polymer Analysis
Aromatic
Dithranol with 0.1 w/v AgTFA inTHF

Polar
DHB in deionized water
Non-polar
Indoleacrylic acid
or DHB in THF, DMF.
Matrix concentration
10
-1
M
Final sample concentration
-4
10
M
Solvents
Sample and matrix dependent. Choose solvents in
which polymer and matrix are soluble.

Preparation
Combine samplematrix (11).
If using metal adduction add in ratio of 2100
to matrix
Dry down quickly under vacuum for even response.
If you allow to air dry, you will see a less even
response during analysis

Crystals
No crystals visible.
If sample position looks glassy or shiny, it may
indicate sample concentration is too high. This
is also indicated by a rainbow effect to the
naked eye.
Analyze polymer samples within one hour of
loading on the sample plate. Some polymer/matrix
mixtures are not stable once they are loaded,
e.g. those loaded in dithranol should be analyzed
with in an hour.
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Polymer Analysis
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Matrix selection
Super DHB (DHB 10 5-methoxysalicylic acid)
Analytes Proteins and glycoproteins Preparation
Solution A 10 g/L DHB in 20 ACN Solution B
10 g/L 5-methoxysalicylic acid in 50 ACN Combine
AB (91)
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Matrix selection
3-HPA (3-hydroxypicolinic acid)
Analytes oligonucleotides gt3500
Da Preparation Prepare saturated solution of
3-HPA in Milli-Q water (50 g/L) and 50 g/L
solution of diammonium citrate. Combine
3-HPANH4citrate 91 .
Note Alternate matrices for oligonucleotides are
THAP (2,4,6-trihydroxyacetophenone) and ATT
(6-aza-2-thiothymine). Desalting is essential.
Do not use HPLC-grade water
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DNA Analysis
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Sample spotting techniques

• Dried droplet
• Crushed crystal
• Thin layer (acetone)
• Sandwich
• Thin layer (nitrocellulose)

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Sample spotting techniques

• Dried Droplet
• Most common method used with all matrices
• Premix sample and matrix solution in 500 ul
  Eppendorf tube, spot 0.5-1.0 µl onto sample plate
  and air dry.
• For small sample amounts, deposit 0.5 ul of
  sample on plate, then add 0.5 ul matrix on top
  mix and allow to dry.

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Dried Droplet Method - Peptides
Sensitivity 50 femtomole digest of bakers yeast
Enolase in CHCA matrix
Reflector Mode Acc. Voltage20000V Grid
Voltage72.5 Guide Wire0.002 Delay time100ns
Counts
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MH
Linear Mode Acc. Voltage 25kV Grid
Voltage90.5 Delay time600ns Guide Wire0.1
M Sinapinic acid
Fragment
M2 Sinapinic acid
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Sample spotting techniques

• Thin Layer (Acetone)
• Use 10 mg/ml CHCA in acetone. Apply 0.5 ml to the
  sample plate to spread and dry.
• Place 0.5 ml sample in 0.1 TFA on top of the
  matrix layer and leave to dry in air.
• If the sample is not acidified then 0.5 ml 0.1
  TFA can be added to the plate, then the sample.
• On-plate washing can be performed after drying.

O. Vorm, P. Roepstorff and M. Mann, Anal. Chem.
66, 3281 (1994)
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Sample spotting techniques

• Sandwich Method
• Deposit matrix on the plate as a thin layer in
  acetone (CHCA, SA) or Methanol (THAP).
• Add 0.5 ml sample in 0.1 TFA, then 0.5 ml
  matrix (in 50 ACN with 0.1 TFA) and dry in air.
  
• Wash with cold 0.1 TFA if desired.

M. Kussmann, et al., J. Mass Spectrom. 32,
593-601(1997).
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Sample spotting techniques

• Thin layer (nitrocellulose) method
• Make up 20 mg/ml solution of NC (BioRad
  Trans-Blot) in acetone, mix 11 with isopropanol.
• Add 20 mg/ml of dry, recrystallized CHCA to this
  solution.
• Deposit 0.5 ml on the sample plate and dry.
• Add 0.5 ml sample on top of the NC/CHCA layer and
  allow to dry.
• To wash, add 1 ml 0.1 TFA, wait 5 sec., blow off
  with compressed air, repeat.

I. K. Perera, J. Perkins and S. Kantartzoglou,
Rapid Commun. In Mass Spectrom. 9, 180 (1995).
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Thin Layer Nitrocellulose Method
Sensitivity 50 attomoles of neurotensin
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Sample spotting techniques

• Crushed crystal method
• Make up a saturated solution of SA in 30 ACN.
• Deposit 1 ml of this solution on the sample plate
  and allow to dry.
• Crush the crystals formed with, e.g., a spatula
  or a foil-wrapped pencil eraser.
• Add 0.5-1 ml sample in saturated SA on top of the
  crystal layer and allow to dry.
• This method may also be used with CHCA

F. Xiang and R. C. Beavis, Rapid Commun. In Mass
Spectrom. 8, 199-204 (1994) R. Beavis and B. T.
Chait, Methods in Enzymology 270, 519-551 (1996).
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Sample Plates
Stainless steel flat plates, 100 spot
Application Proteomics for close external
calibration routines Also easier to see
crystallization of matrix with SS than
gold Stainless steel plates, 100 spot, circles
only Application Easier to locate sample within
a circular moat and flat for close external
calibration 400, 384, 192 -spot Teflon-coated
plates Application Concentrating sample for
increased sensitivity and better location of
sample. Useful in high-throughput
applications. Gold Welled Plates Application
Contains spread of sample and matrix if high
organic solvent e.g synthetic polymers. Also
allows on plate reactions within the well before
drying.
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Voyager 96x2 Sample Plate P/N V700813
Sample Plates
Application 96-well Microtiterplate format with
provision for close external calibrant at each
sample position
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Voyager Biospectrometry Accessories

• Welled Sample Plates
• Gold, 100-well V700401
• Gold, 100-well (no pin, for Voyager built in
  1995 or earlier) V700208
• Flat Sample Plates (Laser Etched)
• Stainless steel, 100-position V700664
• Stainless steel, 100-position (no pin)
  V700665
• Stainless steel, 100-position (numbers and
  circles) V700666
• Stainless steel, 100-position (numbers and
  circles, no pin) V700667
• Special Sample Plates
• Stainless steel, polished blank surface
  V700668
• Stainless steel, polished blank surface (no pin)
  V700669
• Gold, 64-well, disposable, used with disposable
  sample plate holder V503476
• Disposable sample plate holder V700314
• Biacore Chip (holds 4 biochips) V700697
• Membrane, Gels V700698
• Hydrophobic plastic surface, flat, 400-position
  V700699
• 96 2-position, flat, hydrophobic plastic
  surface plate V700813

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