FULL report- Chemistry
Introduction:…the usual, clearly state the purpose first and then explain the chemistry involved … don’t copy the manual or just list calculations
Procedure: …the usual…full account of what you actually did in the lab in the order that you did it, written in past tense and paragraph format (i.e. full sentences) do not just copy the lab manual and change tense
– lab computer printout has absorbance data
-Also include a table of volumes of HNO3, NaSCN and Fe(NO3)3 actually used in the 5 solutions (Vi , Vf and Vused)
Calculations: (see p. 32 in manual)
-show one full set of calculations for one solution…remember to show equations used, labels, units and proper sig figs
– also show calculation of :
average deviation (see p. 8 in manual)
% deviation (average deviation / average Keq) x 100
Discussion: – the usual…
– state the results
– compare to expected & comment
– sources of experimental error
Remember to arrange the sections in the order as listed above and attach the signed carbons to the back
CHEM 3322 – Instrumental Methods of Analysis
Reports should be 1.5 lines spaced. Word and page limits should be maintained.
Abstract: (Maximum 200 words)
Summarize in a concise paragraph the purpose of the report, data presented and major conclusions. What was done, why was it done, how was it done, what were the results and what conclusion(s) can be reported? Analytical abstracts must contain actual values when available. Read a few abstracts from published papers for additional guidance.
Introduction: (1 page max)
- Define the subject of the report: “Why was this study performed?”
- What is the significance and relevance? In other words, why did we do this?
- Outline scientific purpose(s) and/or objective(s): “What are the specific hypotheses and the experimental design for investigation?”
Materials and Methods: (2 page max)
- List chemicals used, any preparation required, who made them (vendor) and their concentration, purity and lot number where appropriate.
- List all equipment used (instrumentation, columns etc.). Listing common equipment like glassware is not required.
- Provide step-by-step procedure with enough detail for the reader to understand the experiment. Make sure to note weights, volumes, and any special glassware and equipment where appropriate.
Results: (Page length as needed)
- Concentrate on general trends and differences and not on trivial details.
- Summarize the data from the experiments without discussing their implications • Organize data into tables, figures, graphs, etc. as needed.
- Provide titles for all figures and tables; include a legend explaining symbols, abbreviations, or special methods if necessary
- Number figures and tables separately and refer to them in the text by their number, i.e. 1. Figure 1 shows that the activity…. 2. The activity decreases after five minutes (fig. 1) Discussion: (2 pages max)
- Interpret the data; do not restate the results
- Relate results to existing theory and knowledge
- Explain the logic that allows you to accept or reject your original hypotheses • Speculate if necessary but identify it as such
- Provide an explanation for poor or unexpected results. Put some thought to what may have caused these results Conclusions: (1 Paragraph)
- Summarize the laboratory exercise and final outcomes Additional Guidance Length Reports are typically 4-8 pages in length including the title, data and references. Rarely if ever below 5 pages and occasionally above 8-10 pages if a significant amount of data/chromatograms are requested. Do not ever exceed 15 pages per report. Double spacing, large font and other ‘creative’ efforts to make the report appear longer are not impressive to the reviewer. Great reports are not necessarily long reports. Brevity in scientific writing is an important skill to master. General Appropriate grammar and readability of reports is required. If you have concerns with your ability to write effectively in English you must seek help from outside of this course. Please let me know if you have any questions. Abstracts for analytical reports should contain actual values/results and should not exceed 200 words. I want to know what you did in the experimental section, not just cut/paste from the lab manual and use of words such as approximately to describe amounts such as volumes and weights. Lab reports must be written in MS Word and submitted electronically through TurnItIn in Blackboard. Please use the convention M8G4Hall (Module #, Group #, Last Name) when naming files. Some guidance on point values and issues to look out for with laboratory reports: Note: This list is not all-inclusive but represents some of the most common errors and resulting point deductions. 1-point deductions: 1-point, Typos/Misspellings/Incorrect language, grammar 1-point, use of colloquial/non-scientific language 1-point, name and module number not provided on report 1-point, proper labeling of figures below and tables above 1-point, proper referencing 1-point, minor technical issues 1-point, incorrect filename convention 3-point deductions: 3-points, Questions must be written out and answered in a defined section of the report Variable Point
Deductions: 1-5points, the manufacturer/model of all instruments as well as solutions prepared or provided must be included in the materials/methods section. Whenever possible organize consumables into tables or bullet points rather than embedded within a paragraph. 3-5points, Moderate to significant technical issues/concerns 3-5points, 200 word limit for abstract. 200 = -3, >250 = -5 3-5points, discretionary. Limited discussion. Lacking pertinent information.
Module 14 – Instrumental Analysis Lab (Jared Auclair, Ph.D.)
Intact Protein Analysis of Ubiquitin, Myoglobin, and Bovine Serum Albumin using Ultra High Pressure Liquid Chromatography Mass Spectrometry Background Biopharmaceuticals are gaining front-page attention with the recent FDA approval of the first biosimilar drug, Zarzio®, a biosimilar version of Amgen’s Neupogen®. A principle component of FDA drug approval is analytical characterization using mass spectrometry. In particular, using protein mass spectrometry to validate the quality of protein drugs. The mission of the Biopharmaceutical Analysis Training Laboratory (BATL) is to train scientists in protein mass spectrometry and then apply those skills to real world problems in biopharmaceutical analysis. Of utmost importance in the analysis of proteins using mass spectrometry is to ensure that there are no clinically significant differences between different lots of protein drugs. Thus, protein mass spectrometry allows us to analyze the intact mass of a given protein along with any given posttranslational modifications that may be present (eg. glycosylation, phosphorylation, amongst others). This information is vital in determining the quality of a given protein. Objective In this laboratory module we will explore a variety of proteins (ubiquitin, myoglobin, and bovine serum albumin). We will cover the preparation and analysis of these samples and considerations that need to be taken into account for proper analysis. In addition, we will modify these proteins with hydrogen peroxide and look for oxygen modifications in the intact mass spectrum. We will analyze the samples by UPLC-MS and interpret the resulting data. Safety Considerations As with all laboratory experiments personal protective equipment should be used including: a lab coat, goggles, and gloves. No open toe shoes or shorts will be permitted in the lab. Experimental Design Considerations Part #1: Preparation of samples for LC-MS analysis Ubiquitin ~ 8 kDa; Myoglobin ~15 kDa; BSA ~ 66kDa Part #2: Ubiquitin, Myoglobin and Bovine Serum Albumin Analysis: unmodified and modified. Each lab group should analyze an unmodified protein (either Ubiquitin, Myoglobin, or BSA) and a modified form of the same protein. Part #3: Interpretation of data. Critical Instrument Parameters Experimental Design Each student will conduct each of the sections with the supervision of the instructor. Data will only be shared amongst the members of the lab group (2-3) present on the day of the BATL visit. Each student will report on their own data and the data obtained by their lab partner. Data will not be shared amongst all members of the lab group. (1.1) Preparation of the Intact Protein: Desalting 1. Take a Millipore Amicon Ultra-4 with a 10,000 Da molecular weight cut-off and fill it with 4 mL 10 mM ammonium bicarbonate, pH 8.0. 2. Add 50 µL 10 mg/mL protein solution (Ubiquitin, Myoglobin or BSA) to the amicon. Cap the tube and invert 3 times to mix your sample. 3. Spin for 20 minutes at 3500 RPM in the swinging bucket centrifuge. Note: the maximum speed for this rotor is ~3800 RPM. 4. After the 20-minute spin, remove the filter ensuring the sample stays in the filter part of the amicon and decant the liquid that passed through the filter into the sink. 5. Add 4 mL of HPLC water to the amicon tube (top filter part) and resuspend by inverting. 6. Spin for 20 minutes at 3500 RPM in the swinging bucket centrifuge. 7. Repeat steps 4-6, 3 more times. In an effort to save time, during Step 6 (centrifugation), move to Step 1.2 below. 8. After the last spin the final volume of sample your sample should be 50 µL. As the sample loss is usually minimal, we’ll assume the concentration remains at 10 mg/mL. 9. Prepare a 1 mg/mL stock of your protein by diluting a 10 mg/mL stock 1:10: add 2 µL of the 10 mg/mL stock to 18 µL of HPLC water. (1.2) Preparation of the oxidized sample: Prepare your oxidized protein by incubating with hydrogen peroxide. Incubate a 10 mg/mL protein sample with 10 mM hydrogen peroxide for 1 hour at room temperature. Incubate 10 µL of 10 mg/mL protein with 1 µL of 100 mM hydrogen peroxide. After the one-hour incubation spin in the microcentrifuge for 5 minutes at 14,000 RPM to pellet any precipitate. Prepare a 1 mg/mL stock of your protein by diluting a 10 mg/mL stock 1:10: add 2 µL of the 10 mg/mL stock to 18 µL of HPLC water. (1.3) Calibration of the Waters Xevo G2S Q-Tof: Sodium cesium iodide clusters will be used to calibrate the Q-Tof using direct injection from the “C” injection valve. Using the instrument parameters from above, we will calibrate the instrument in the intact protein mass range of 500-4000 m/z using the instruments calibration sequence. (2.1) Ubiquitin, Myoglobin and Bovine Serum Albumin Analysis: unmodified and modified: Each individual will place 10 uL of their 1 mg/mL unmodified protein sample in an autosampler vial with a preslit cap and 10 uL of their 1 mg/mL modified protein sample in a separate autosampler vial with a preslit cap. Then add them to the sample manager and take note of which position you placed your tubes. We will run your samples using a C18 reverse phase column and the gradient below. Note: buffer A is water/0.1% formic acid and buffer B is acetonitrile/0.1% formic acid. Each student will add their samples to the sample table using a sample name to include their initials, date, and protein. The injection volume will be 2-3 uL per sample (2-3 ug total protein injected). (3.1) Interpretation of data: Data will be manually interpreted where possible. This will include a discussion of the charge state distribution, determining individual charge states, and how to calculate molecular mass using this information ([m/z * z]-z). In this case, the instructor will use previously collected data from SOD1. In addition, we will discuss determining a theoretical mass for your protein using uniprot.org and expasy.org. Also, we will use the maximum entropy 1 function to have the computer software deconvolute our data and translate into the mass domain (Da). Results, Calculations and Discussion 1.) What is the importance of sample preparation? Why did we desalt your sample? 2.) Calculate the theoretical molecular weight of your protein to two decimal points. 3.) Identify the charge state distribution associated with your unmodified protein? Use this information to calculate the molecular weight of your protein; select the most prevalent peak and any other smaller (modified) peaks. How does this compare to your maximum entropy deconvoluted molecular weight? How does this compare to your theoretical molecular weight? What modifications do you see (in daltons; eg +32 Da, etc.)? What might they be? 4.) Answer the same questions from number 3 for your modified sample. Did the hydrogen peroxide treatment change your protein in any way? Do you think oxidation of your protein will affect its function? 5.) If your total ion chromatogram wasn’t well resolved (you had overlapping proteins eluting at the same time) what might you do to get better resolution? 6.) What role does protein mass spectrometry play in modern day biotechnology? Report Data LC/MS data (2 total chromatograms and selected MS data) should be included in the report for all sections of this lab.