Sample Preparation for Mass Spectrometry Analysis
Good sample preparation is essential for good data.
Sample types analyzed
- gel bands, stained with Coomassie, silver, fluorescent stains. Note that some silver stains are incompatible with mass spectrometry analysis (see the protocols suitable for sequencing).
- proteins in solution
- tissue samples
- Laser Capture Microdissection tissue samples (BTRF)
- FFPE tissue-see review M.B. O’Rourke and M.P. Padula, Biotechniques 60:229-238 (May 2016) doi 10.2144/000114414 See synopsis
For all samples
- Enter a service request and sample information in iLab.
- Give sample tubes unique names
- 1.5 ml microfuge tubes preferred
- Bring to room 1105 Pinn Hall
- Samples in tubes can be placed in freezer in racks for MALDI or ESI analysis
- Place sample sheet in tray on freezer.
- If submitting gel bands, bring the whole gel and a picture showing the bands or spots to be analyzed.
- If shipping samples from outside the University of Virginia, address to:
Dr. Nicholas Sherman
Biomolecular Analysis Facility, Box 800741,
University of Virginia Health System,
Pinn Hall room 1105
1340 Jefferson Park Avenue
Charlottesville, VA 22903
Samples for protein identification
- Bring the whole gel, not just bands
- Detergents need removal
- Only some silver stains are compatible with mass spectrometry; one we have success with is from
MALDI Sample preparation
Samples can be run by staff, or by researchers under the open access scheme. To use the open access scheme, users need to contact Dr. Sherman and then arrange training with Dr. Shannon or Park.. Users are expected to provide their own sample plate and supplies (sinapinic acid and cyanohydroxycinnamic acid matrices are provided by facility).
The facility is usually open 8:30 a.m. to 5 p.m.
- 10 pmole/µL or more desired, 5 µL or more desired
- SDS incompatible
- Other detergents cause problems but special procedures may get data
- molecules <600 MW may be obscured by matrix
- telling us the purpose of the experiment is helpful
Laser Capture Microdissection samples
- successful sample preparation takes skill
- we have seen good results when prepared with Expression Pathology kit
- Laser Capture Microdissection is available in the
Keratin is found in almost all samples because humans shed pieces of skin. Common recommendations to reduce contamination include:
- wear nitrile gloves (latex may introduce latex proteins)
- do not lean over gels, skin particles may fall from your head introducing keratin
- for gels, use commercial precast gels or commercially prepared reagents if casting your own
- keep solutions in closed containers to exclude dust
- keep labware covered or enclosed to exclude dust, and close pipet tip boxes
- work in an area cleaned of dust before hand, preferably a laminar flow hood
- avoid long sleeves and wool when handling samples; long sleeves funnel hairs and skin flakes towards the sample
- use tubes that have been kept sealed from possible dust
- when opening a tube containing a sample, blow with clean air to remove dust from the tube
- do not use gel staining trays which have been used for blocking reagents for Western blots