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Sample Preparation

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Sample Preparation Guidelines

** Important - To minimize human keratin contamination, we recommend:  

  • Handle gel plugs/slices inside clean laminar flow cabinet
  • Wear clean gloves
  • Use clean tubes for storage

Sample concentration requirements

  • Peptides (< 5 kDa): minimum 1 pmol/µL
  • Proteins (>=5 kDa): minimum 5 pmol/µL
  • Enzyme digests: minimum 1 pmol/µL

General guidelines for all sample types

  1. Always use MS compatible solvents.
  2. Minimize salt concentration in samples.  Avoid phosphate buffer, detergents, salt and ammonium salts or derivatives of organic amines. [Solvent & Buffer]
  3. Provide estimated sample concentration.
  4. Use low-binding tips and tubes.
  5. Use high quality plastics (polymer has been found sheding from poor quality plastics and interfering with downstream MS.  Most famous brandnames seem to be OK.)

Samples containing high salt or detergent content may be cleaned up with C18 ZipTip at additional cost.

Service Type Sample Type Specific Guidelines

Full Service
Protein Identification

(WITH sample
preparation)

Gel plug
(1D or 2D)

MUST use MS compatible staining.

Excise stained gel bands or spots of interest by using clean razor blades or disposable pipette tips.

Store gel pieces in clean microfuge tubes at room temperature.

Undigested pure protein
(in solution)

Keep salt level as low as possible

NO detergent in samples

Prepare samples in solution of 10 mM Ammonium Bicarbonate at pH 8.0.

Store samples in clean microfuge tubes at -20ºC.

* Keep samples on ice during shipment.

Protein Identification

(WITHOUT sample preparation)

Digested peptides

MUST be in 3–5 µL of salt-FREE and additive-FREE solvent for optimum results

Freeze-dry or dissolve samples in 3–5 µL of 0.1% Formic acid: 50% Acetonitrile or 0.1% Trifluoroacetic acid: 50% Acetonitrile.

Store samples in clean microfuge tubes at -20ºC.

* Keep samples on ice during shipment.

* Provide spectrum if sample is separated by LC.

Digested Samples
spotted on MALDI plate

Spot samples on a regular 384 MALDI plate.

Prepare an Excel file containing sample names and spot locations. [service charges]

Mass Determination

Protein, other biological materials or small molecules
(please enquiry)

MUST be in 3–5 µL of salt-FREE and additive-FREE solvent for optimum results

Freeze-dry or dissolve samples in 3–5 µL of 0.1% Formic acid: 50% Acetonitrile or 0.1% Trifluoroacetic acid: 50% Acetonitrile.

Store samples in clean microfuge tubes at -20ºC.

* Keep samples on ice during shipment.

* Provide spectrum if sample is separated by LC.

Digested Samples
spotted on MALDI plate

Spot samples on 384 MALDI plate.

Prepare an Excel file containing sample names and spot locations. [service charges]

Additional ZipTip® Desalting Service
(included in Full Service)

Samples may contain salt, detergent [FAQ]

Freeze-dry samples: dissolve in 15-20 µL of 0.1% Formic acid or 0.1% Trifluoroacetic acid.

* Ensure NO Acetonitrile residues in samples.


Solvents and Buffers

Acceptable To be Avoided

Water: Double-deionized such as Milli-Q® grade 18 mΩ

Organic Solvent: Methanol, Acetonitrile

Acids: Formic acid, Acetic acid, Trifluoroacetic acid

Bases: <=10 mM ammonium acetate, <=10 mM ammonium bicarbonate, <=10 mM ammonium formate, <=2% triethylamine

Water: HPLC grade water (contains varied amount of salts)

Buffers: Phosphate Buffer

Salts: NaCl (common in buffers or solvents from cation- or anion-exchange purification), ammonium salts or derivatives of organic amines, e.g. 2M guanidine, glycerol, sucrose, ethanolamine

Detergents: > 0.01% non-ionic detergent, e.g. TWEEN20, Triton X-100, CHAPS, NP-40 or anionic detergent e.g. SDS.

* A detergent concentration of 0.1% variably affect signal response of >= 60% dependent upon the detergent used. At 1%, most detergents eliminated the protein signal.