Analyzing Large Protein Complexes by Structural Mass Spectrometry

The overall goal of the following experiment is to characterize large protein assemblies using structural mass spectrometry. This is achieved by preparing gold-plated capillaries for nano flow electro spray ionization. As a second step, the sample is prepared and the mass spectrometer is calibrated for high mass measurements.

Next, experimental conditions are optimized in order to maintain protein complexes intact in the gas phase. MS and tandem Ms.Spectra are then acquired. The MS Spectra showed charge states corresponding in mass to the intact complex while tandem MS experiments yielded association pattern of monomers and stripped complexes.

Hi, I'm Naam Kiba from the Laboratory of Miron in the Whiteman Institute of Science, And I am Isaac Ky.I'm also from the laboratory of michelon. Today we would like to show you the basic procedure for analyzing large protein complexes by applying structural spectrometry, And we use this procedure in our laboratory to characterize protein complexes to define your stock geometry, composition, and networks of interaction. So let's get started.

Analysis of noncurrent bound protein complexes is performed by air spray immunization technique using as emme plus or a qua capillaries. This capillary C can be pulled to fine tip of about one micron diameters and then coated by conducting material like gold. These capillaries are commercially available as you can see in here.

However, it's much more cost effective to use. The glass capillaries, eh, which can be than cool and coated to the ready to use state cooling, is performed on this needle pooling device and coating on this eh coating device. To begin this procedure, attach two strips of a double-sided adhesive pad to the bottom of a Petri dish, keeping the strips two centimeters apart.

Place a glass rod in the center of one of the pads. The adhesive pad holds the capillaries in place and the glass rod supports the prepared capillaries and keeps the tips from breaking for the capillaries. Used Boris silicate glass capillaries with one millimeter outer diameter and 0.78 millimeter in a diameter.

Insert one capillary into the needle, clump the capillary gently in place and adjust its position so that it lies in the center of the heating Filament. Tighten the clamps gently until the capillaries held firm at both ends. Pull the capillary using the predefined program shown here.

However, remember that the process of programming the puller is one of trial and error. Until an acceptable tip shape is obtained, every capillary pulled gives rise to two final shaped capillaries. Remove the pulled capillaries from the instrument and inspect the tips, discarding any that are deformed or broken.

Use blunt positioning tweezers to place the capillaries in the Petri dish. The base of the capillaries attached to the adhesive pad and the upper part leans on the glass rod with a tip pointing up. Continue pulling the capillaries until the Petri dish is full.

About 80 capillaries fit into a 10 centimeter diameter dish. Next, coat the capillaries with gold. Insert the plate into the gold sputter coter.

Make sure that the gas supply is chosen according to the manufacturer's instructions and activate a predefined coating cycle. The coating is repeated three to six times until the capillaries are evenly golden. Once the capillaries are ready, proceed to calibrate the mass spectrometer.

Most of the experiments conducted on MultiPro complexes are performed by a al Electrospray quadruple time of flight mass spectrometer like this. Inupt from waters, which is modified for high masses. Before loading the protein sample, calibrate the mass spectrometer using caesium iodide.

First, prepare a 100 milligrams per milliliter solution of caesium in purified water, Caesium is used for high mass calibration. As the singly charged salt clusters extend over a wide mass range from 393 mast to charge ratio to well over 10, 000. Using blunt tweezers, take a coated capillary from the Petri dish and load two microliters of the cesium solution into the capillary.

Using an einor gel loaded tip. Slide the solution towards the tip of the capillary, either manually or use a spin down adapter. Insert the capillary into the capillary holder and adjust the capillary in such a way that the tip is approximately 10 millimeters away from the edge of the holder.

Place the capillary under an optical microscope and use sharp AA type tweezers to trim the tip. It is important to validate that there are no air bubbles within the capillary that could block the flow and that the capillary is not clogged. In addition, make sure that the gold coating is not stripped from the capillary.

If so, replace the capillary or trim the tip and remove the uncoated section. Next, connect the capillary holder to the nano flow ESI interface. Rotate the XY, Z stage backwards to avoid damaging the capillary and push the stage into its activated position.

The capillary should be placed at a one to 10 millimeter distance from the cone orifice. Apply capillary voltage and low nano flow pressure until spray is initiated. Then try to reduce the nano flow pressure to a minimal value.

Optimize the signal intensity by adjusting the location of the XYZ stage, the capillary voltage, the nano flow pressure, and the diss solvation gas flow. To detect a wide mass range of season peak series, the accelerating voltages should be optimized. The parameters used here are capillary 1.3 to 1.7 kilovolts sample cone 80 to 150 volts, an extraction cone, one to three volts to optimize the transmission of high mass ions, which require gentle desal conditions.

The backing pressure is raised to five to six millibar in the initial vacuum stage between the source and the analyzer. To raise the backing pressure, carefully reduce the conductance of the source vacuum line to the scroll pump by partially closing the isolation valve while monitoring the effect on signal intensity. When setting up the mass spectrometers complete, collect about 30 scans at one scan per second at a master charge ratio range of between 1000 to 15, 000 after acquisition calibrate the time of flight using the appropriate calibration table.

Now that the calibration is complete, the protein sample can be loaded. The sample is prepared before calibrating the mass spectrometer low micromolar concentrations are required. If necessary, concentrate the sample using centrifugal ultra filtration devices.

Verify that the protein complex is not absorbed to the device membrane before use. By checking the volume and UV absorbance only volatile solutions may be used for NESI often making buffer exchange necessary. A micro centrifuge gel filtration column can be used to remove all traces of salts, buffer molecules, or any other non-volatile adducts.

Instead, suspend the sample. An aqueous ammonium acetate solution. This critical step determines the quality of the spectra.

Repeat the buffer exchange up to three times with minimal dilution, less than a factor of 1.3 per device until maximum exchange is achieved. If both concentration and buffer exchange are required, these may be done together using centrifugal ultra filtration as just shown in the beginning of this section. Next, load the sample as shown for the caesium iodide calibration and initiate spray.

Start by initial optimization of the spray until the signal is detected. Use the equation for average charge state shown in which M is the mass of the complex. In Daltons, however, the exact position of the charge states is protein dependent.

To prevent complex dissociation, do not heat the iron source. Either switch the heater off or keep the temperature below 40 degrees Celsius. Vary the capillary position, sample cone and extractor voltages.

To maximize iron transmission, a possible starting point is cone voltage, 100 volts extractor cone, one volt and capillary voltage 1.5 kilovolts. Check the resulting change in the spectra. Optimize these parameters in combination with a nano flow pressure to improve deification and strip off residual water and buffer components.

Increase the bias voltage in the gas pressure in the collision cell. Typical bias voltages are within the range of 10 to 100 volts with the trap gas flow of one to 10 milliliters per minute. Perform this carefully to prevent association of the complex, adjust trap and transfer collision energies.

Often higher voltages usually within the range of 10 to 30 volts are required for transmission of high mass ions. At this point, it is important to avoid collision induced dissociation of the complex as shown here. After a stable signal is reached, it has recommended that the nano flow pressure and capillary voltage be decreased to minimal values while maintaining stable spray.

After collecting several scans, proceed to identify the components of the complex by tandem mass spectrometry. Once an optimum and stable signal has been obtained for the protein complex, select a precursor ion set the mass center in isolation width. Generally a low mass resolution of 12 and a high mass resolution of between 13 and 15 use a wide mass range to detect the high mass low charge dissociation products and then reduce it to the desired values To perform ms.

MS dissociate the precursor iron By increasing the collision energy abbreviated CE and the pressure on the collision cell increase either the trap or transfer CE gradually in steps of 10 to 20 volts and elevate the collision gas pressure to zero to five milliliters per minute. Monitor changes in the spectra until optimal activation conditions are reached. High activation energy may induce the dissociation of one or more subunits from the intact complex and clarify the interaction affinities of different subunits.

Select more than one charge for ms MS analysis. In the case of overlapping components, acquiring a set of tandem MS Spectra assist in resolving the charge series of the different populations. Moreover, higher charge states dissociate more easily compared to lower charge states superimpose the MS and ms.

Ms spectra to validate the isolation of the chosen precursor ion. In addition to the characterization of the intact complex by MS and ms. MS generates smaller sub complexes and solution under mild denaturing conditions and characterize these as well.

The MS and ms MS analysis of sub complexes form the basis for defining the subunit architecture of the complex for partial disruption of subunit subunit interactions gradually add organic solvents up to a concentration of 50%or change the pH of the solution by adding ammonia or formic acid up to a concentration of 4%Finally, to determine the masses of the individual subunits that compose the complex, it is important to acquire spectrum under denaturing conditions. This can be carried out using zip tip C four with a 25 to 75 water aceto nitrile ratio with 1%formic acid as the elucian solvent with all the data collected process and analyze the results as detailed in the accompanying written protocol. Before loading the protein complex sample, the mass spectrometers calibrated using cesium iodide.

The series of equally spaced peaks marked in red extend over a wide range from a master charge ratio of 393 to well over 10, 000. They're assigned to singly charge salt clusters of the general composition of the caesium plus complex. Additional signals between the major peaks marked in black are caused by double and triple charge species of the series caesium two plus complex and the caesium three plus complex.

Increasing the pressure at the initial vacuum stage is essential for detecting the high mass clusters. The effect of pressure on the high mass peaks is demonstrated here with pressure read backs of 1.2 and 5.3 millibar. The mass spectrum for the higher pressure is shown expanded here.

Here is an example of mass spectrometry of a lectin variant complex giving rise to a charge state distribution between 3000 and 5, 000 master charge ratio. However, due to an adequate desal of the ions, the peaks are broad. Increasing the bias voltage from four volts to 15 volts, which brings about an increase in accelerating conditions, causes the stripping of residual water and buffer components yielding a highly resolved spectrum, the measured mass corresponds to a pentameric complex.

The plus 15 charge state was then selected for tandem MS analysis. The increase in collision energy causes the release of a highly charged monomer centered at 1, 664 master charge ratio and a stripped tetrameric complex in the range of 5, 000 to 8, 000 mass charge ratio. The lectin variant complex was also examined under denaturing conditions leading to complex association.

In this way, the accurate mass of the individual subunits making up the complex can be determined. We have just shown you how to acquire MS and tandem Ms Spectra of high mass ions. When doing this procedure, it is important to properly prepare your sample and use only volatility buffers the compatible with mass spectrometry.

So that's it. Thank you for watching and good luck with your experiments.