Sample preparation

Sample PreparationSample Preparation

Voyager Training Class

Sinapinic Acid Proteins >10kDa

a-Cyano-4-hydroxy-cinnamic acid (CHCA)

Peptides<10kDa

2,5-Dihydroxybenzoic acid (DHB)

Neutral Carbohydrates,Synthetic Polymers

“Super DHB” Proteins,Glycosylated proteinsOligonucleotides

HABA Proteins,Oligosaccharides

3-Hydroxypicolinic acid

Matrix selection

Vacuum dried

Milky, amorphous

Air dried

Irregular crystals in ring

DHB Crystallization

3 HPANeedles in fan-like arrangement

around outer edge of sample well

THAP

Overlapping shinglesnon-uniform shape

DNA Analysis

- cyano- cyano Sinapinic acidSinapinic acid

Rounded Rhomboid-shaped

Appearance of Matrix

Sample Plates

1 mm diameter

Ability to load 1µl sample and matrix

Application: Enhanced sensitivity by concentrating sample; useful in

high throughput methods

Advanced Sample PreparationAdvanced Sample Preparation

Voyager Training Class

Sample Dilution/Concentration

Note: highly dilute samples can be concentrated by Speed-Vac or Solid Phase Extraction.

Compound Concentration0.1 to 10 pmol/µL

10 to 100 pmol/µL

Peptides and proteins

Oligonucleotides

Polymers 100 pmol/µL

Dilute samples to the concentrations shown in the table below.If the sample concentration is unknown a dilution series may be needed to produce a

good spot on the MALDI plate.

Drop dialysis cleanup of Enolase

Yeast Enolase (47 kDa) in 8 M urea was dialyzed for 1 hr on a Millipore membrane.

After

Before

•ConditionCondition the ZipTip with 10 µl of acetonitrile (ACN), then 10 µl of 50% ACN/0.1% TFA, then 2 x 10 µl of 0.1% TFA.

•LoadLoad the sample onto the ZipTip by pipetting 5-10 µl sample up and down several times and discarding the liquid.

•WashWash C18 tip with 3 x 10 µl of 0.1% TFA to remove salts.

•EluteElute the sample from the ZipTip with 30-70% ACN or elute directly into the matrix (e.g. CHCA in 50% ACN/0.1%TFA); minimal volume of ~3 µl can be used.

Procedure for using C18 ZipTips

Use of the C18 ZipTip

1.1. Sample Concentration and Buffer RemovalSample Concentration and Buffer Removal

Dilute samples can be concentrated by adsorbing analyte from multiple 10 l aliquots into the ZipTip and eluting out into a small volume, effecting a 10- to 50-fold concentration.

Mild conditions (e.g. 0.1% TFA) will retain peptides and proteins on a ZipTip but remove common buffers and salts such as: 2M NaCl, 100mM Phosphate,

8M Urea, 6M Guanidine or 50% Glycerol

For more detailed information on ZipTips, click here

C18 Efficiently Removes digestion Buffer

-40000

-20000

0

20000

40000

Co

un

ts

1000 1500 2000 2500 3000 3500 4000 4500 Mass (m/z)

C18 Prep in PBS/Urea/NaCl

Standard Prep in PBS/Urea/NaCl

Analysis of a peptide map of IgG HC digest containing phosphate, NaCl, urea and DTT at 0.1 mg/ml digested with endo Lys C.

Protein Sequencing of Myoglobin using In-Source Decay

-Le

u/I

le(1

13

.27

1)

-Ph

e(1

47

.29

5)

-Th

r(1

01

.41

4)

-Gly

(56

.23

4)

-?(2

34

.91

2)

-Glu

(12

8.7

76

)

-Th

r(1

01

.18

3)

-Le

u/I

le(1

13

.11

2)

-Glu

(12

9.2

4)

-Gln

/Ly

s(1

27

.78

6)

-Ph

e(1

47

.78

3) -A

sp

(11

4.9

58

)

-Gln

/Ly

s(1

28

.38

7)

-he

(14

6.

P4

96

)

-Gln

/Ly

s(1

28

.17

4)

-His

(13

6.7

46

)

-Le

u/I

le(1

13

.48

8)

55

75

.10

2

3500 4000 4500 5000 5500

Ref:V.Katta et.al.Anal.Chem.1998,70,4410-4416

ProteomicsProteomics

Voyager Training Class

In-Gel Digest Fundamentals

• Handling the gel and slices

• Washing and destaining

• Enzymatic Digestion

• Peptide Extraction

• Concentration/Cleanup

• MALDI-TOF Analysis

In-Gel Digest MethodMALDI-TOF AnalysisMALDI-TOF Analysis

•Acquire a good spectrum in reflector mode with a method optimized for high resolution in 800-3000 Da range. Calibrate with internal Trypsin peaks T7 (842.5099) and T4 (2211.1046) if present, otherwise use close external calibration.

•Alternatively, samples can be spiked with dilute Cal Mix 1 or 2 (approx. 1:500 in the matrix) for internal calibration. Finally, samples can be internally re-calibrated with known peak masses from a good Protein Prospector MS-Fit hit.  •If spectrum is poor due to contaminants or low peptide concentration try cleanup and/or concentration of the remaining sample with ZipTip C18

•

Nanotechnology - carbon clusters