LC-MS guidelines Orbitrap Exploris™ 480 Mass Spectrometer + Dionex Ultimate 3000 HPLC system
Liquid Chromatography-Mass Spectrometry is a technique used to get information about the peptides present in a sample (identity, sequence, relative quantitation, chemical modifications, etc). After preparation, samples are introduced into an MS vial (there are several types, brands, etc). The vial is deposited in an autosampler from where it is injected into the LC sample loop and introduced into the LC-column. The column tip lies inside the MS source, from where the charged sample’s droplets are volatilized and go inside the MS via the Ion Transfer Tube (ITT). The positively charged components on the sample (generally peptides) are analyzed in the orbitrap. On a typical tandem MS run, charged peptides are directed to the CID/HCD cell for fragmentation. The masses of the fragments produced in this step are used for sequence and ID information.
Sample Preparation
There are several protocols that can be followed in order to get samples ready for the MS. Mostly, they require the use of high purity reagents and low-bind protein tubes in order to get samples as clean as possible for analysis. The last step of sample preparation is usually to speed-vac them to dryness (overnight is usually accepted). Samples are then resuspended in solvents containing 0.1 % formic acid (FA), which is needed to get positively charged peptides (acid-base equilibrium). Various solvents can be used to resuspend depending mostly on the nature of the samples and applications. As a general rule, the solvent used to resuspend the samples should not be less polar than the most polar solvent of the LC-run gradient (e g, if the LC-run begins by using 96.9:3.0:0.1 water:MeCN:FA, the solvent used to resuspend the samples should not contain more than 3.0 % MeCN).
Autosampler distribution
Dionex Ultimate 3000’s autosampler has three racks that can hold 40 MS-vials each. Additionally, there are 18 extra spaces to place bigger sample vials. Apart from the samples, the autosampler should always contain a vial (can be a big one) with solvent A (99.9:0.1 water:FA) to inject when blanks need to be ran; and a vial containing a solution of 0.1 micrograms/microliter of Pierce standard HeLa digest resuspended in solvent A. This solution must be run periodically (see HeLa standard) to ensure LC-MS system performance is up to standards.
LC-MS solvents
Solvents used during the runs can be found on top of the LC unit. Only LC-MS quality solvents can be used. After preparation, solvents need to be sonicated/degassed for at least 20 minutes. Before starting any new set of runs be sure that the solvent bottles are not empty and that there is enough solvent to complete the runs. Solvents used are four and their composition varies upon application. Generally, solvent A is 99.9:0.1 water:FA, solvent B is 99.9:0.1 MeCN:FA, needle strong wash 40 to 60 % MeOH with 0.1% FA. A mixture 1:1:1:1 IPA:MeOH:MeCN:water is also commonly used. Finally, needle weak wash is usually the same as solvent A. Solvents must be changed as often as possible in order to avoid bacterial growth, changes in their composition due to differential volatilization, etc. NEVER touch a solvent bottle without gloves to avoid contamination sources. Avoid the use of plastics.
LC columns
Analytical columns come in different lengths and can be Taylor made to use different types of silica (depending on the application). For regular bottom-up proteomics, 50 cm length c-18 silica modified columns that are commercially available are often chosen. Analytical columns can be connected to trap columns to protect them from unwanted contaminants that may damage them.
MS sources
Ionization sources also come in different types. There are two kinds of sources in the laboratory: sources used to calibrate, and the ones used for the LC-MS runs. They are easily distinguishable and can be exchanged (see calibration).
Running a set of samples
Place the vials containing the samples on the autosampler, making sure that there are no bubbles inside, especially at the bottom part. Open the exploris software and either create a new sequence file or add one line per sample to an existing sequence. Specify the name of each sample (e. g. IP_complex_buffer_conditions_replicateN), the position of the sample in the autosampler, the volume you wish to inject, the instrument method (choose the one that better adjust your needs, if you wish to create a method for your runs and do not know how to, ask for technical support), and the path were you want your RAW files to be stored. Make sure there is enough space in the disk for all your raw files, otherwise the acquisition will stop. As a rule, replicates are run one after the next with a blank in between two sets of replicates to avoid carryover from one sample to another. Before starting a new set of samples, always run a blank and a Hela standard.
Hela standard
To ensure optimal instrument performance, run Hela standards periodically. Inject 500 ng of Hela digest and run them using the specified method. Once the search is ready, check that the number of protein group IDs are above 2500 and the max BPI is above 1E+09. If any of these conditions is not met, stop your queue and ask for technical assistance. Always run a blank after a Hela.
Ion Transfer Tube (ITT)
The ITT needs to be cleaned and replaced for a clean one if the signal of your samples is getting lower or, ideally, at least once a week. To replace the ITT, follow the steps shown in this video https://vimeo.com/197201795 . To clean the ITT, sonicate with LC-MS qualiy IPA for 30 minutes, then air dry and store safely in the tube provided ad hoc.
Calibration
The instrument needs to be calibrated once a week. Put the instrument on Stand-by and replace the LC-MS source for the calibration source. ASK FOR HELP IF YOU ARE UNSURE HOW TO DO IT. Turn the instrument on and allow it to get back to normal. Fill the pump syringe with calibration solution. Make sure it is the right one. Connect the tube that comes out of the syringe to the source and initiate the syringe flow with a 10 uL/minute flow until you see the calibration mix on the spectra. Once you see all the peaks, change the flowrate to 5 uL/minute Check signal stability (TIC). Once it is stable for 200 runs, initiate the calibration process as signaled by the instrument. Once the calibration passed, you can change the source back and continue running samples. ALWAYS calibrate after ITT replacement and not before. ALWAYS run a Hela after calibration.