Proteomics and Metabolomics Laboratory

Frequently Asked Questions

  1. What is LC/MS?
    This abbreviation stands for Liquid Chromatography/Mass Spectrometry. LC or HPLC is used for separation of substances based on specific physical properties such as hydrophobicity or charge.  The mass spectrometer is the detector which measures mass to charge ratio (m/z) which can be used to determine molecular weight. This technique is considered the most sensitive and accurate for the detection of substances.
  2. What is LC/MS/MS?
    This abbreviation stands for Liquid Chromatography on-line Tandem Mass Spectrometry. The MS spectra contains enough information to determine molecular weight but does not give information regarding structure.  Due to this, many mass spectrometric methods utilize tandem mass spectrometry or MS/MS.  In these experiments, the molecule of interest is isolated and subjected to fragmentation.  The resulting fragment ion spectrum, MS/MS spectra, contains information on the structure of the compound.  For peptides, this spectrum can be used to determine the amino acid sequence of the peptide allowing for protein identification.  For small molecule MS, the combination of the specific parent mass and the unique fragment pattern is used to selectively monitor and quantify the compound of interest in a robust, versatile and high-throughput manner. Compared to LC/MS, LC/MS/MS method is much more specific and sensitive for quantitation of small molecules (100-2000 Da).
  3. Can endogenous compounds (<2000 Da) and drugs be quantified in biological samples like plasma/serum using LC/MS or LC/MS/MS?
    Yes.
  4. Can novel compound in biological samples be identified using LC/MS/MS?
    Yes, if the quantity of the compound is enough in biological samples.
  5. How much protein do I need for identification?
    Any band that can be detected with Coomassie staining contains enough protein for a successful analysis. In my experience, this requires approximately 1 pmol of protein, but the exact amount is dependent on the protein. The protein must be visualized to be cut from the gel.
  6. What about analysis of silver stained bands?
    The analysis of silver stained protein bands is certainly possible with mass spectrometric methods. The mass spectrometry is amply sensitive for the analysis of fmol amounts of protein so it is fundamentally able to sequence silver stained bands. However, two significant practical problems must be addressed and can destroy the effectiveness of the experiment. 1) Glutaraldehyde fixation modifies proteins in a manner that makes them impossible to digest. This reagent simply cannot be used in the staining procedure. 2) The background created by both protein and non-protein contaminants of the gel will mask the signals produced by peptides derived from the protein of interest. These contaminants are certainly also in Coomassie stained gels, but the inherently larger amount of analyte protein overcome the problems these species create. As a result, the ultimate success of experiments sequencing silver stained bands is generally dependent on absolute cleanliness and care at every stage of the experiment, including the sample preparation and electrophoresis step. Our success rate for these experiments is around 70%.
  7. What is the turnaround time?
    Our goal is to be able to provide preliminary data within two weeks and a written report within three weeks.
  8. How pure does the sample need to be?
    Any protein that can be cut from a gel can be sequenced, no matter how many additional bands are present in the gel. There will always be some concern that a band is composed of more than one protein, but this should not affect the ability to detect and sequence peptides produced in the digestion.
  9. Can I identify post-translational modifications?
    Post-translational modifications can be identified by mass spectrometry; however these experiments generally require more protein than the identification experiments. This is especially true for modifications that are sub-stoichiometric in nature. One of the inherent difficulties in these types of analysis is identifying a low abundant modified peptide present in a complex peptide mixture. Several different strategies can be sued to increase the likelihood of success in these experiments including large amounts of protein, increasing the stoichiometry of the modification reactions, using targeted analysis and multiple proteases.
  10. Can the amounts of my protein or post-translational modification be quantified?
    Relative quantities of proteins and post-translational modifications can be determined in these experiments. In order to achieve this, an internal standard must be utilized as a normalization factor. Fortunately, in most samples an unchanging tryptic peptide inherent in the sample can serve as an internal standard.