Electrospray Ionization - Mass Spectrometry
Electrospray Ionization (ESI)
The ionization technique transforms ions of proteins and peptides from a liquid solution to a gaseous state before mass spectrometric detection. ESI Mass Spectrometry (ESI-MS) has high sensitivity and robustness, thus making it well-suited for non-volatile molecules and complex sample solutions.
Liquid Chromatography Mass Spectrometry (LC-MS & LC-MS/MS)
An HPLC separates the peptides and proteins (in liquid solution) according to chemical properties like hydrophobicity, size, and charge. The components are released from the stationary phase into the mobile phase and subsequently detected and analyzed in the mass spectrometer (MS). If the mixture is subject to tandem mass spectrometry (MS/MS), the first MS selects precursor ions by their mass (m/z), fragments them, and then detects them as product ions in the second MS.
Combining LC-MS with UV detection is also possible, thereby achieving UV trace chromatogram, MS ion current chromatogram, and MS spectra. This method is known as UV HPLC analysis.
A variant of LC-MS/MS with flow rates in the nanoliter/min range. NanoLC-MS/MS has a very long analysis time due to low flow rates. The system can thus only run smaller sample amounts but has higher sensitivity than the conventional LC-MS/MS setup.
SWATH mass spectrometry is a data-independent acquisition (DIA) technique for detecting and quantifying all detectable compounds in a complex sample. Only the SCIEX TripleTOF instrument performs this analysis.
Multiple Reaction Monitoring (MRM LC-MS/MS)
Technique especially suited for quantification of specific, single proteins of particular interest (HCPs). MRM has high sensitivity and specificity, thus making it perfect for targeted analysis of a set of low-level proteins, even if they are only ppm-level abundant.