Guilty As ‘Charged’ – Identifying the Protein Using Mass Spectrometry.
At Peak Proteins we routinely use protein mass spectrometry methods to fully characterise and understand the proteins we have produced in our protein expression systems. So why do we do this and what additional information do we gain, in addition to SDS-PAGE gel analysis and analytical size exclusion as part of our quality control? In this article we will outline the basics of protein mass spectrometry, the system we use and the information we can obtain from both intact protein and peptide mapping techniques. At Peak Proteins we use a Sciex Exion LC coupled to a Sciex X500B mass spectrometer. The X500B is a benchtop QTOF (Quadrupole Time of Flight) mass spectrometer. It is made up of a series of quadrupoles and a TOF detector.
Mass spectrometers basically measure the mass/charge(m/z) ratio of ions to identify and/or quantify molecules in simple and complex mixtures. Protein or peptide samples are loaded into the mass spectrometer in liquid form via the LC and then vaporized and ionized by the ion source. This is known as electrospray ionisation or ESI. The charged molecules accelerate through the quadrupoles and TOF tube. Individual ions based on their m/z travel towards the detector and the time it takes to hit the detector is proportional to the m/z. This entire process is performed under an extreme vacuum to remove gas molecules and contaminating non-sample ions, which can collide with sample ions and alter their paths. A chromatogram is generated to show the ions that have been detected in the run; this is known as a total ion chromatogram (TIC). At Peak Proteins we use Sciex BioToolKit and BioPharmaView software programs to analyse the data and organise them by their individual m/z values and relative abundance.
The Sciex X500B mass spectrometer at Peak Proteins is routinely used to analyse intact protein mass along with peptide mapping to determine sequence coverage of proteins. So, what does each method tell us?
For most proteins we know the theoretical mass of the protein that we have expressed so use Intact Mass Analysis to confirm that we have purified the correct protein. However, by analysing any mass additions we can also find out if the protein has any post translation modifications (PTM) such as phosphorylation, glycosylation, oxidation and biotinylation to name a few. We can also determine if the protein is processed correctly (good news) or degraded (not good news!). So, a lot of very precise information and with a much quicker turnaround time than running and staining a gel….
To run an intact protein mass sample, we use ESI-LCMS analysis, where protein samples are loaded in liquid form and the protein is ionised and passed through the X500B to the detector. To enable ESI-LCMS, the sample is loaded onto the Sciex Exion LC and a 5-minute reverse phase gradient is used with buffer A as 0.1% formic acid and Buffer B as 0.1% formic acid 100% acetonitrile. A Phenomenex Jupiter 5um, C4, 300A 50×2.1mm column is routinely used. This captures the protein and then elutes when the acetonitrile concentration increases. The flow from the column is passed into the Sciex X500B mass spectrometer collecting data in positive ion mode. The resultant TIC is deconvoluted using Sciex BioToolKit software to provide an accurate mass of the protein and/or potential PTMs.
Peptide Mapping on the other hand is performed using ESI-LCMSMS analysis where is addition to detecting the mass of the peptide ions, the sequence information can also be deduced. MSMS is performed in the second quadrupole of the X500B, where peptide parent ions are selected and then subjected to CID (collision induced dissociation) with the use of gas and voltages. The bonds break within the parent ion to generate ions which can then be measured at the same time . The ion pattern can be compared to a theoretical pattern to find the sequence of the peptide.
Generally, protein bands from a Coomassie stained gel are used for peptide mapping, or if the protein is very pure, a liquid digest can be performed. The samples are reduced with DTT to break disulphides and the reduced cysteines alkylated with iodoacetamide, to form a carbamidomethyl cysteine. The protein is then digested with trypsin, and/or other proteases. The resultant peptides are extracted and loaded onto the Sciex Exion LC and a 10 or 20-minute reverse phase gradient is used with buffer A as 0.1% formic acid and Buffer B as 0.1% formic acid 100% acetonitrile. A Phenomenex Lunar 1.6um, PS C18, 100A 150×2.1mm column is used. The flow from the column is passed into the Sciex X500B mass spectrometer collecting data in positive ion mode. Peptides bind and then elute off the column depending on their hydrophobicity. MSMS data is collected using an Information Dependent Acquisition (IDA) method where up to 10 or 20 MSMS are collected per scan. The data is searched against the above sequence using BioPharmaView and also against Swissprot using Mascot.
Performing peptide mapping analysis allows us get a greater understanding of specific changes to the protein such as whether any degradation seen is from the N or C- terminus of the protein, or confirmation of engineered mutational changes. It can also confirm the specific amino acid sites that have been post-translationally modified or can determine the compound binding sites of covalent compounds. If immunoprecipitation has been used to pull down the target protein, peptide mapping can also identify specific protein binding partners or even contaminating proteins that we may want to remove by further purification steps.
Even the amount of additional information that can be obtained versus simple SDS-PAGE gel analysis, it’s clear why having the equipment and expertise in protein mass spectrometry is crucial for us to provide quality analysis and assurance to our clients that we have indeed produced the proteins they requested!
Have a look at our protein mass spectrometry service page to find out more about how we can help you analyse your protein samples.
