HRMS and Targeted MS services
HRMS is the acronym for high resolution mass spectrometry, and it is often used to describe a process that involves the use of a mass spectrometer with high mass accuracy to measure the mass of a compound. The obtained value is used to confirm the formula of a known molecule or generate potential formulas for unknown molecules. Isotopic patterns are also used to further validate the formula generated based on exact mass. The accepted tolerance for the mass error depends on the weight of the molecule under analysis. As a general guideline, errors below 5 part-per-million (ppm) are accepted for mass values > 200 Da. For molecules smaller than 200 Da, a value of 3 mDa is adequate in most cases. The MSF offers HRMS services through our Synapt XS platform
Targeted MS indicates methods to quantify known molecules in an unambiguous manner. This generally requires a combination of different separation and filtering dimensions, and it is often accomplished with triple quadrupole (QqQ) instruments operating in multiple reaction monitoring (MRM) mode. When in MRM mode, a triple quad is operated with the first quadrupole (Q1) acting as a mass filter centered around the m/z of the ion of interest (the parent ion) with a user defined tolerance. The second quadrupole (Q2) is operated as a collision cell to fragment the ions that fall within the mass window allowed by Q1. The third quadrupole (Q3) operates similarly to Q1, with a mass window centered around a specific fragment associated with the molecule of interest. When combined with the separation provided by liquid chromatography, a targeted LC-MS/MS experiment identify the signal of a molecule by combining retention time, parent ion mass and specific fragment mass for unbiased quantification. This approach can be achieved with other type of instrument as well, although not at the speed achievable with a QqQ, and the MSF offers this service through our Synapt XS and our amaZon platforms.
Description: this type of analysis is aimed at determining the exact mass of the components of a sample without a column separation. The exact mass measurements can be used to calculate theoretical formulas and compare them to the user provided formula for a given compound. Mass errors are usually kept below 5 ppm by using internal calibration. This analysis can be performed in flow injection analysis (FIA) or with a column separation (LC-MS). LC-MS allows for the determination of the mass spectrometric purity of the sample as well.
Amount needed: 20 µL at a concentration of 10 ppm. The concentration refers to the mass of the whole sample, not just the component one wishes to detect.
Type of samples: any ionizable molecule.
MSF protocol available: yes
Degree of difficulty: low
Notes: While this analysis is commonly performed on our Synapt XS platform, we can achieve similar results on our rapifleX MALDI platform as well if we have an internal standard (or if we can use matrix peaks as calibration points).
Description: this type of analysis utilizes the TOFMRM mode (Synapt XS) or the MRM mode (amaZon), where the mass spectrometer isolates a mass window of a few Daltons at the time based on a user provided list and fragments it. The resulting ions are used to generate the output signal similarly to parallel reaction monitoring on a trapping instrument. This approach is designed to target one molecule at the time and requires optimization of both chromatography and fragmentation conditions to obtain the maximum sensitivity possible. Limits of detection in the part per billion range are generally achieved.
Amount needed: variable, depending on the type of sample and the target compound(s).
Type of samples: Cell, tissue, organoids, plants, biofilms etc.
How many replicates: the minimum is 3, but the number is dictated by the variability of the target(s) under analysis.
MSF protocol available: Yes
Degree of difficulty: low
Gradient length: generally, 5-10 minutes.
Notes: the TOFMRM methods may or may not utilize ion mobility. The maximum acquisition rate for the Synapt XS is 30 Hz, which limits the number of transitions that can be monitored when compared to dedicated instruments such as triple quadrupoles. The acquisition rate of the ion trap is similar, although the mass precision is lower.
HRMS analyses are performed with Waters MassLynx, which offers tools for elemental composition determination. Users are provided with platform-independent raw spectra as well.
Targeted analysis is performed with Waters MassLynx (Synapt XS) or DataAnalysis (amaZon), both of which provide tools for visualization and integration of the acquired data. Further calculations are provided with Microsoft Excel.