Peptide Mass Fingerprinting of Egg White Proteins

Abstract

Use of advanced mass spectrometry techniques in the undergraduate setting has burgeoned in the past decade. However, relatively few undergraduate experiments examine the proteomics tools of protein digestion, peptide accurate mass determination, and database searching, also known as peptide mass fingerprinting. In this experiment, biochemistry students digest a protein mixture from egg white using the enzyme trypsin; liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC–ESI-TOF-MS) separates the resulting peptides and determines their accurate masses. Instrument software is used to match these peptides to the sequences of known egg white proteins, obtained from an online source. Students then use online protein database search software to match the peptides to the protein and score the results.

Protein Identification by Database Searching of Mass Spectrometry Data in the Teaching of Proteomics

“…The importance of mass spectrometry for proteomics makes it a relevant topic to be taught to undergraduate students. A number of initiatives with that purpose are found in the literature, some of which are very comprehensive, including hands-on activities from protein separation by electrophoresis to identification to mass spectrometry. − However, many of the proposed classes or courses focus on mass spectrometry itself, giving little or no attention to the principles of database searching. Although database searching is usually mentioned, instructors treat it as a self-explanatory procedure that deserves little attention.…”

“…Although database searching is usually mentioned, instructors treat it as a self-explanatory procedure that deserves little attention. In many papers, it is described as little more than “plugging in the data and clicking the “OK” button”, with no exposition of the underlying theory. ,, Some instructors provide only superficial indications for how to set the search parameters, , while others reduce the result interpretation to merely looking at the protein with the highest score . In a recent paper, the authors cite online material that can guide students along broad and comprehensive theory, as well as provide valuable exercises, for the learning of protein identification by mass spectrometry.…”

Hands-On Electrospray Ionization Mass Spectrometry for Undergraduate Biochemistry Students: Peptide Identification by Ladder Sequencing

“…Cohen et al (2005) developed a laboratory exercise in which students discover two given proteins and their origin using ESI-MS for total mass determination, MALDI-TOF for peptide mass mapping, and ion-trap tandem (fragmentation) mass spectrometry for sequence analysis . Alty and LaRiviere (2016) integrated liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) into the undergraduate setting to identify tryptic peptides originating from proteins in egg white, comparing the experimental results with known peptide sequences of egg white proteins …”

Identification of Proteins Using Four Methods of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

“…8 An egg-white protein mixture was digested with trypsin; the resulting peptides were analyzed using LC-ESI-TOF-MS and then matched to the known peptides in egg-white proteins. 9 Modern proteomic research most often deals with mixtures of proteins from known sources, and reviewers of such work expect that PMF is insufficient for identification: at least one peptide (preferably more than one) from each protein must be sequenced to clearly establish the identity of each protein. 3,10,11 This laboratory experiment builds upon previous work listed above and addresses the lack of teaching hands-on experiments regarding protein/peptide sequencing using MALDI.…”