CHEM525 Experiment 8: MALDI-TOF MS analysis of FPLC purified Pectate Lyases Introduction In this experiment, we will be using matrix-assisted laser

desorption ionization mass spectrometry to obtain an accurate mass of the pectate lyase proteins you have purified. This mass will be the final piece of the puzzle necessary to confirm the identity of the proteins. MALDI-TOF mass spectrometry is a robust technique that is highly amenable to biological sample analysis due to its soft ionization and tolerance for the presence of salts (<200 mM) in the samples to be analyzed. It is also extremely accurate and capable of analyzing both small peptides and very large proteins. For this laboratory, we will analyze aliquots of your purified, concentrated protein samples on a mass spectrometer in positive ion, linear mode. The mass spectrometer will be calibrated with two protein standards that bracket the mass range we are looking at, thereby ensuring that we obtain the most accurate mass possible. Reagents/Equipment Needed Purified pectate lyase samples, concentrated to 1 mg/mL 1mg /mL Sinapinic acid in 70% acetonitrile/0.1% TFA (MALDI matrix) 2 mg/mL Bovine serum albumin (MW: 66431.10 g/mole) (Mass standard) 2 mg/mL Apomyoglobin (MW: 16952.27 g/mole) (Mass standard) 100 well stainless steel MALDI-TOF sample plate Applied Biosystems Voyager DE STR Mass Spectrometer Procedure 1) Prepare the matrix solution by adding 1 uL of each of the MALDI mass standards to 30 uL of sinapinic acid solution. 2) Add 1 uL of the matrix solution to 2 adjacent spots on the MALDI plate. Do not use the spots on the edge of the plate! This is a precaution to prevent your sample from being contaminated or even destroyed in case someone mishandles the plate. 3) Quickly add 1 uL of matrix solution to the first spot and 1 uL of your purified protein solution to the second spot. This will give you two spots that consist of 2 uL of solution. 4) Allow the spots to dry. 5) The plate will be loaded into the mass spectrometer and sample data will be collected. Be certain to record the instrument parameters at time of use for inclusion into your lab report. 6) Once a good spectrum is obtained, save the data file in the CHEM525 directory with a filename that you will recognize. Record the filename in your lab book for future reference.

7) With assistance from the instructor, manually calibrate the mass spectrum of the matrix only spot (the first spot). Print the resulting spectrum as your negative control. Manually calibrate the data from the spot containing your protein and print the spectrum. Precautions/Note on good technique: Do not handle the plate with bare hands, nor spot the samples out with your face directly over the plate. Our skin constantly sheds keratin and this rain will fall onto the plate and contaminate the results. Requirements for lab report Since there is not much experimental work to do in this experiment, you will have no excuse for not focusing on perfecting the Materials and Methods section of your writeup. In the discussion section for the lab report, you will need to discuss the results of the MALDI-TOF MS experiment. The mass of mature pectate lyase C is 37696.78 g/mole and that of pectate lyase E is 38171.34 g/mole. If the observed masses in the calibrated mass spectra do not match these values, then you will need to discuss the discrepancy. The most likely differences will be attributable to protease activities.