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

Abstract

Mass spectrometers are ever-increasingly powerful, user-friendly, and affordable. Thus, the addition of mass spectrometry experiments into the undergraduate laboratory curriculum is now both feasible and an effective tool to introduce students to relevant instrumentation. Here an experiment demonstrating the use of a highresolution electrospray ionization mass spectrometer (ESI-HRMS) for the identification of peptide sequences and the differentiation of isomeric peptides was developed and implemented in a biochemistry laboratory course for third-year chemistry major undergraduate students. This lab activity was a first introduction to mass spectrometry for undergraduate biochemistry students. As a prelab assignment, students were tasked to predict the mass-to-charge ratios and the fragmentation patterns for six given peptides using an online fragmentation prediction tool (Protein Prospector's MS-Product tool). Students then analyzed two unknown peptides using ESI-HRMS. The theoretical and experimental results were compared to reveal the identity of the two unknown peptides. The student success rate of recognizing the unknown peptide sequences was 87.5%. This laboratory experiment provided students with hands-on experience using a research-grade ESI-HRMS instrument to solve a bioanalytical problemspecifically, the identification of the primary structure of a peptide of unknown sequence. Learning outcomes were evaluated for this experiment, which showed student understanding of peptide sequencing using mass spectrometry.

Real-Time Monitoring of Reactions by Headspace Sampling under Ambient Mass Spectrometric Conditions

“…The utility of mass spectrometry in the undergraduate laboratory has been demonstrated through experiments involving, for example, essential oils in citrus fruits (GC-MS), 5 the analysis of caffeine (ESI), 6 environmental pollutants (GC-MS), 7 gasoline constituents (GC-MS), 8 as well as by studying primary kinetic isotope effects (GC-MS), 9 the measurement of gas-phase basicities of amino acids (ESI), 10 and peptide identification by ladder sequencing (ESI). 11 Reactions between aptamers and proteins such as tubulin have also been studied (ESI). 12 Less frequently, MALDI-MS has been used to monitor reactions, e.g., solidphase peptide synthesis.…”

An Interdisciplinary Experiment within Chemistry: Natural Product Composition Analysis Using a Miniature Mass Spectrometer

“…Actually, instrumental analysis and its experiments are often compulsory courses for science and technology majors such as chemistry, biology, environmental science, food science, and pharmaceuticals in colleges and universities. Spectroscopic and chromatographic analyses are now widely incorporated into undergraduate laboratory training. − Mass spectrometry, however, has not yet been popularized in routine undergraduate laboratory teaching, although it has been incorporated at an increasing number of universities. − This may be due to the characteristics of commercial MS instruments. Traditional commercial MS instruments are large-scale precision equipment, characterized by large size, high price, precision manufacturing of components, high requirements for the cleanliness of the experimental environment, and high instrument maintenance costs.…”

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.…”