Peptidi e proteine quantificazione utilizzando Automated Immuno-MALDI (iMALDI)

The overall goal of this automated immuno-MALDI assay is to quantify peptides and intact proteins in complex biological fluids in an accurate and sensitive manner. This method can help answer key questions in the quantitative proteomics field, such as quantify protein levels for diagnostics or for monitoring a patient’s response to treatment. The key advantage of this method is that it can quantify protein levels very sensitively and accurately from complex samples.

This technique allows the diagnosis of diseases at an early stage because the immuno-enrichment step of the assay allows quantitation of low abundant protein and peptide biomarkers. Though this method can provide insight into plasma renin activity, it can also be applied to other disease-related studies, such as quantification of cancer-related proteins for prognosis, diagnosis, and treatment management. To begin this procedure, thaw human plasma samples in a room temperature water bath for five minutes.

Then, place the samples on ice until completely thawed. Next, manually transfer 200 microliters of each plasma sample to separate wells of a 1.1 milliliter deep well plate. Centrifuge the plate for 10 minutes at two degrees Celsius and 3, 000 rpm.

Then, use the Bravo to serially dilute a 500 femtomole per microliter Ang-1 NAT standard solution to prepare six calibrator solutions with chicken egg white albumin. Following centrifugation, pipette 200 microliters of each calibrator to a well and 125 microliters of generation buffer to the sample plate. Using an automated liquid handling system, mix 125 microliters of plasma supernatant or 125 microliters of the chicken egg white albumin solution with 25 microliters of angiotensin I generation buffer in a new 1.1 milliliter deep well plate.

Now, automatically transfer the solutions to a 96-well plate with three replicates per solution and 34 microliters of solution per well. Then, incubate the plate at 37 degrees Celsius for three hours. For conjugation of the antibody with protein G beads, first transfer 78 microliters of previously prepared bead slurry to a 1.5 milliliter tube.

Wash the beads seven times with one milliliter of 25%acetonitrile in PBSC and three times with one milliliter of PBSC, using a magnetic stand to pellet the beads between each washing. After washing the beads, resuspend them in 110 microliters of PBSC and add 110 microliters of anti-angiotensin I antibody. Mix the beads and solution by pipetting.

Then, incubate the beads at room temperature for one hour while rotating at eight rpm. Following this, wash the beads three times with one milliliter of PBSC. Then, resuspend the beads in 1, 100 microliters of PBSC.

Now, manually transfer the bead solution to a 96-well plate. Then, have the Bravo aliquot the beads to the same 96-well plate. After the three-hour angiotensin I generation period, place the incubation plate on ice for 10 minutes to terminate the generation of angiotensin I.Automatically dilute a stable isotope standards peptide stock solution 100-fold with PBS buffer.

Further, automatically aliquot the stable isotope standard peptide dilution to a 96-well PCR plate. Transfer 10 microliters of the diluted peptide solution to each well of the incubation plate. Mix the diluted peptide solution with the plasma samples or the chicken egg white albumin solution.

Automatically add the sample or standard solutions to the bead solutions to the incubation plate and mix. After incubating one hour, automatically wash the beads three times with five millimolar ammonium bicarbonate solution. Use a magnet to pull the beads to the bottom after each wash.

After the last wash, add 10 microliters of the ammonium bicarbonate solution to each well to resuspend the beads. Automatically transfer seven microliters of the bead slurry onto a MALDI target plate with a spot size of 2, 600 micrometers. Once the beads have dried, automatically add two microliters of previously prepared HCCA matrix solution from the matrix well onto each sample spot on the target plate.

Following this, analyze the sample spots with a MALDI-TOF instrument using positive reflector mode. Perform internal calibration, data smoothing, and baseline subtraction with the vendor-specific software. The automated iMALDI procedure for measuring angiotensin I allows for high-throughput analysis of a large number of samples with an intraday coefficient of variation below 10%Representative spectra obtained by measuring angiotensin I in human plasma samples are displayed here.

Correlation of PRA values from 188 patient samples obtained with the automated iMALDI assay with PRA values obtained using a clinical LC-MS/MS procedure is shown here. The two methods have a correlation coefficient of 0.98, and the difference between the slopes may be due to the use of different internal standards or different antibodies. The linear range of the assay and the assay precision are displayed here.

Once mastered, this technique can be performed in approximately five hours. While attempting this procedure, remember to properly adjust the tip head and the liquid handling speed. After this procedure, additional methods can be used to confirm primary aldosteronism in patients, including oral salt loading or saline infusion tests.

The automated iMALDI technique allows the specific and accurate high-throughout quantitative screening of biological samples, including patient samples, for disease-related proteins or peptides. After watching this video, you should have a good understanding of how to perform an automate iMALDI assay for mass spectrometry-based protein and peptide quantification. Don’t forget that working with biological samples, such as human plasma, is extremely hazardous, and the necessary safety precautions must always be taken while performing the procedure.