This module is an introduction to biological mass spectrometry with an emphasis on proteomics, the study of proteins, particularly their structures and functions. The first section provides key background information on proteins and proteomics. The second section describes the use of electrospray ionization to determine the molecular weight of a protein using an ion trap mass analyzer. The third section shows how to identify an unknown protein using peptide mass fingerprinting with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). The fourth section describes shotgun proteomics and tandem MS experiments for de novo peptide sequencing.
Each section includes some brief explanatory text with accompanying reading questions that emphasize student comprehension. The text and accompanying reading questions are intended to be completed as a pre-class assignment so that students have the background information needed to work on more difficult questions during the class period. Alternatively, the reading questions could be completed at the start of the class period. Each section also includes discussion questions and activities that are intended for in-class work in small groups. The instructor’s manual gives more detailed suggestions for how different portions of the module might be used and the amount of class time needed to complete each section.
Information on how to use this module
What prior knowledge do students need before beginning this module?
It is helpful if students have had at least one class period on the basics of mass spectrometry before starting this module. For example, students should understand that analytes must be ionized for mass spectrometric analysis so that the molecules may be separated by m/z. No prior knowledge of proteomics, specific ion source, or mass analyzers is needed because these topics are covered in the module.
The information in Section 1 is a necessary prerequisite to Sections 2-4, but Sections 2-4 are independent of each other. An individual instructor could complete one or two of these sections without the others and in any order. Students who have taken biochemistry may not need Section 1A, which is an introduction to amino acids, the peptide bond, and proteins.
How much time is required in and out of class to complete each section?
Section 1: Proteins and proteomics
Class Period 1: (Section 1A What is a protein?) Reading questions should be completed prior to class. Discussion questions can be answered in small groups during class.
Class Period 2: (Section 1 B Proteomics) Reading questions should be completed prior to class. It is best to summarize and discuss the answers to this set of reading questions at the start of class. During class, distribute the following comparative proteomics paper and have students work in groups to answer the discussion questions.
Comparative proteomics of oral cancer cell lines: identification of cancer associated proteins, Karsani et al, Proteome Science, 2014, 12:3. doi:10.1186/1477-5956-12-3 (Open access journal)
Section 2: Calculating the molecular weight of a protein from its electrospray ionization mass spectrum
Class Period 1: Reading and video questions should be completed prior to class. Discussion questions can be answered in small groups during class. (If all questions are completed during the class period, then two class sessions are needed.)
Section 3: Identification of a protein: Peptide mass mapping
Class Period 1 and 2: Two full class periods are suggested for the reading and discussion of MALDI and TOF-MS (Sections 3A-B).
Class Period 3: Section 3C Reading questions should be completed prior to class. Discussion questions can be answered in small groups during class.
Class Periods 4 and 5: Section 3D Reading questions should be completed prior to class. Students will need a computer to access the maxtrix science website and mascot database. It is best if students download an excel file of the mass list and then they can simply copy and paste the m/z values into the search query. Students will explore how changing search parameters affects the protein score (statistical significance that a protein has been correctly identified). The varying protein scores must be interpreted.
A series of homework problems accompanies Section 3D. These are meant to be assigned out of class but could also be used during another class period.
Section 4: Sequencing peptides and identifying proteins from tandem MS (MS-MS) data
Class Period 1 and 2: If the reading and video questions are completed prior to class, the discussion questions can be completed in one to two class periods, depending on whether the groups work through sketching fragment ions and calculating masses together or divide the work among group members. To save time, the instructor may allow students to use the Institute for Systems Biology’s peptide calculator. The Challenge Question may be assigned as homework or worked in class during a class session. If readings, videos, and discussions will all occur in class, at least three class periods are recommended.
What is the difference between reading questions, video questions, and discussion questions?
Reading questions and video questions are short answer questions that are relatively straightforward. Individual students should be able to answer these questions on their own without group discussion. Reading questions are based on reading the text portion of the module, and video questions are based on linked videos. Students could be assigned to read and view videos before coming to class. This will ensure that students have the requisite background knowledge to discuss the more open-ended and challenging discussion questions in small groups during class. It is best to briefly re-cap the answers to the reading questions at the start of the class period.
Alternatively, students could complete the reading and video questions independently during class; this works especially well for shorter sections like the introduction to proteins (Section 1). Discussion questions should always be used in-class where students discuss the answers in groups of 3-4 students. A good strategy is to have each group discuss a question for several minutes while the instructor circulates to answer questions, correct misunderstandings, and monitor discussions. Then the instructor can optionally call the class together as a group to report out answers and for large group discussion before moving on to the next question. For the time-of-flight portion (Section 3B), there is an accompanying slide show that includes animations to be shown as a wrap-up to discussions.