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Mass Spectrometry Facility

Sample Preparation

In Gel Digestion

The SOP-MSF (School of Pharmacy-Mass Spec Facility) aims at catering to researchers in identifying proteins from 1D and 2D gels using the current state-of-the-art mass spectrometers for proteomic analysis. We are eager to use this expertise to assist you in your studies using mass spectrometry. The methods available to ID proteins in our facility are quite sensitive and can be used to identify proteins in the 100-500 ng range for bands detected by Zinc, SYPRO Ruby or Coomassie Staining.

The following In-gel Digestion protocols may be followed for MALDI-TOF MS Analysis of:

• SDS-PAGE Gel Separated, Coomassie-Stained Proteins
• In-Solution Digestion of Proteins

MALDI Mass Spectrometry

Peptide Mass Fingerprinting (PMF)

Enzymatic digestion of proteins, (in solution or in-gel) followed by MALDI-MS of the resulting peptide mixture leads to a peptide mass fingerprint (map), which is characteristic for the protein present. Using this procedure, an unknown isolated protein can be enzymatically digested by a sequence specific protease, such as trypsin. The resulting mass map measured with MALDI-MS is compared with the calculated masses of all tryptic peptides that can theoretically be produced from sequences corresponding to all the proteins in the genomic database of the organism under study. The protein yielding the best match between theoretical and experimental mass values is identified.

Sample Spotting: 5ul of acidified digest is mixed with equal proportion of MALDI matrix (10mg alpha-cyano-4hydroxy cinnamic acid (HCCA) in 1ml of a mixture of 50% Acetonitrile and 50% 0.1% TFA). The solution is thoroughly mixed and 0.5-1ul is spotted on the MALDI target. Care is taken to prevent keratin contamination by the use of fresh samples, a laminar flow hood and gloves.ha-

Standards used: Peptide Calibration Standard (1000-4000Da) from Bruker Daltonic GmbH (Leipzig, Germany) Part #:206195 is used for external calibration. The peptides in this standard cover a mass range from 757 to 3147 Da. Where improved accuracy is needed, autolysis peaks (842.509, 2211.104) from porcin trypsin (Sequencing Grade Modified Promega Trypsin Catalog # V5111) are used for internal calibration.

Obtaining Sequence Information: From the Peptide Mass Fingerprint (PMF) spectrum, peptides in the mass range of 1000-2000 Da that have a clean base line are selected. Tandem Mass Spectrometry (MS/MS) is performed using PSD (Post Source Decay) on the precursor mass, by employing FAST (Fragmentation Analysis and Structural TOF) method offered by Omniflex.

Protein Identification: The monoisotopic mass maps of tryptic peptides generated, are searched in databases (NCBI nr, MSDB, Swissprot) using MASCOT http://www.matrixscience.com/search_form_select.html search engine. For PMF as well as PSD mass maps,  BIO TOOLS (Software from BrukerDaltonic GmbH, Germany) is used for protein identification.

Protein MW Determination

The conditions used for Protein Molecular Weight determinations by MALDI-TOF-MS may vary with molecular weight. Please ask for details as to how your data was acquired from Contact SOP-MSF.

Samples may be delivered to the SOP-MSF using specific guidelines and tips  as provided below.

In some cases it will be necessary for sampling and analysis protocols to be established in discussion with the SOP-MSF prior to analysis or sample submission. If in doubt Contact the SOP-MSF.

Users of the SOP-MSF Analytical Services agree to the following:

• An appropriate acknowledgement of the involvement of the SOP-MSF will appear in any publication involving work in the SOP-MSF.
• If the SOP-MSF is involved in assay development and/or validation work for the project, co-authorship is required with the appropriate member(s) of the SOP-MSF.
• A copy (or full citation details) of any publication involving work in the SOP-MSF will be sent to the Director of the SOP-MSF within one month of it appearing in the literature.

Submission of an analysis request form provides your agreement to the above conditions.

Sample Submission Guidelines

It is preferred if samples are submitted neat or dried down, typically ug amount is all that is needed.

Biological samples are handled differently and require different conditions. Salts, buffers and surfactants suppress ionization and need to be removed for mass spec analysis. When possible, it is advised to dialyze the sample into nanopure or at least distilled water prior to submission. We also recommend using  several de-salting techniques for proteins and oligonucleotides available commercially. List all salts, buffers and detergents the protein or oligonucleotide is in, or has recently been in. For optimal results, please discuss your project with Dr. Peter Swaan prior to submission. Certain classes of proteins need to be handled differently than others, the more we know about your system, the better the data. Protein samples for MALDI are prepared in 50% acetonitrile 50% water with 0.1% TFA. Slightly acidic conditions are necessary to facilitate protonation.

We cannot run samples that are in aromatic solvents like benzene, toluene, or solvents such as DMSO and DMF.

Electrospray and MALDI samples are prepared in Methanol, Acetonitrile, water, THF, chloroform. We will acidify the sample with acetic or formic acid unless you indicate the sample is acid sensitive.

Turn around times vary between 1 day to a week depending on sample load, instrument availability and type of analysis. We will email you when the results are ready to be picked up.