In order to break a protein down into peptides, there are a few steps that need to be followed, such as denaturation, reduction, alkylation, and enzymatic digestion. This is still true for performing Peptide Mapping on the ZipChip, however, we have developed techniques to make this process as straight-forward and rapid as possible. Our Peptide Mapping protocol includes detailed steps that allows for a sample to be ready for analysis within two hours as opposed to the several days most protein digestion protocols call for.

Our Peptide Mapping specific protocol includes guidelines for sample preparation, system suitability testing, mass spectrometer method parameters, and ZipChip separation method parameters. These guidelines tend to generate robust and accurate data, but every mass spectrometer is different and there might be some slight modifications that need to take place for optimal data collection. This is usually very minimal, even with more complex samples such as a protein digest.

A run time of 10-15 minutes is very common for running a Peptide Mapping analysis of a protein or mAb using ZipChip. Compare this to one or more hours on an LC-MS setup, and the speed benefit becomes very apparent.

Digesting a protein into smaller peptides allows for more detailed post-translational modifications to be recorded. PTMs, such as deamidation, Met Oxidation, Trp Oxidation, Isomerization of Aspartic Acid, Glycosylation, Succinimide Intermediates, or Dioxidation, are all PTMs that have been recorded using the ZipChip coupled to a high resolution mass spectrometer.

Usual sequence coverage for a digested protein or monoclonal antibody (mAb) when separated on a ZipChip is between 95-100%. In contrast, the sequence coverage using an LC-MS system is usually in the low 90%. The reason is that smaller peptides (such as di or tri peptides) tend to not be retained on the stationary phase of an LC, thus not being properly separated and recorded in the mass spectrometer. Since the ZipChip microfluidic chip utilizes an open capillary to separate analytes by charge and size, this is not an issue, and small di and tri peptides do show up in the data, resulting in generally higher sequence coverage of a protein compared to LC-MS.

Yes! The ZipChip utilizes your mass spectrometer’s data collection software, so the output format would be similar to any other separation done using that mass spectrometer as the analyzer. Data analysis software such as Skyline, Proteome Discoverer, MaxQuant, Mascot, MultiQuant, Analyst, Proteoscape, Biopharma Finder, Biopharma ComPass, or any other software capable of quantifying peptides, can be used to process data generated by ZipChip coupled to a mass spectrometer.

There are two major benefits of performing Peptide Mapping on the ZipChip over running it on a Liquid Chromatography – Mass Spectrometry setup. First, the ZipChip separation is extremely fast. A Peptide Mapping analysis that can take hours on an LC-MS system takes minutes on the ZipChip. Second, smaller peptides (such as di or tri peptides) tend to not be retained on the stationary phase of an LC, thus not being properly separated and recorded in the mass spectrometer. Since the ZipChip microfluidic chip utilizes an open capillary to separate analytes by charge and size, this is not an issue, and small di and tri peptides do show up in the data, resulting in generally higher sequence coverage of a protein compared to LC-MS.