Protein and Nucleic Acid Facility

MALDI and LCMS

Service offered: MALDI-TOF mass analysis of the peptide mixture, database searches and protein identification:

The facility's Applied Biosystems Voyager-DE PRO MALDI-TOF mass spectrometer can be used for the analysis of peptides, small molecules, and whole proteins.

One of the major uses of the instrument is analysing peptides from protein digests to identify the protein. A band or spot can be cut from a 1D or 2D gel, the protein digested in-gel, and after Zip-Tip cleanup, the peptides are analyzed with the mass spectrometer.

Peptide Mass Analysis:

1. The sample plate with up to 100 spots is inserted in the mass spectrometer
2. A laser is applied to individual spot thus ionizing molecules of the matrix which in turn transfers a proton to the peptides.
3. Peptides are accelerated through the flight tube under vacuum and in most cases in a reflector mode, which basically makes the flight path longer than the actual tube.
4. Peptides arrive at the detector based on their mass to charge ratio (m/z).
5. Using calibration peptides, the actual masses of the peptides are assigned.

Data base search and Protein Identification:

All the masses that represent peptides from the original protein (in other words, masses present in control samples where no protein was present are ignored) represent the fingerprint of the protein in question. By searching a mass database for protein fingerprints, the protein is identified if known. If we are dealing with an unknown protein, further identification becomes necessary among which is peptide sequencing of selected peptides by post source decay (PSD) or Collision induced dissociation (CID).

Data Provided: Actual mass profile (chromatogram) and data from Mass search (protein identity). Extra data will include PSD and CID (only upon request)

Turn-around time: 5-7 working days

Cost: On-campus (Protein identification) $200.00

Off campus (protein identification) $400.00

Service:  Nano-LC/MS/MS analysis of peptides and peptide fragments.

The Protein and Nucleic acid facility houses an LTQ LC/ MS mass spectrometer from Thermo-Fisher.  This instrument offers the high capacity of a linear ion trap and very high levels of accuracy and sensitivity.  Using the LTQ we are capable of identifying proteins in the fmol range.  The LTQ is also used in the identification posttranslational modifications.  One of the major applications of this instrument is the identification of total proteomes in mixtures such as serum, urine, cellular fractions, etc.  
Samples are first  digested with an appropriate protease, most commonly used is trypsin .  Peptides are extracted from the gels, if gels were used, and Zip-Tip cleaned.  Peptide mixtures are then applied to the nano-LC/MS where peptides and peptide fragments are identified.  The data is searched against appropriate databases for protein identification

Data base search and Protein Identification:

All MS/MS data are searched against appropriate databases and proteins are identified.

Data Provided: A visualize program is supplied with each sample analyzed.  The purpose of this program is to simplify understanding of the data. It is a great application where the data can be exported to a number of other applications that can be used in grant applications, posters, etc.

Turn-around time: 5-10 working days

Cost:

On-campus: $350/sample
Off campus: $500/sample