(LC) Liquid Chromatography:
Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the the components in a mixture.
A column holds the stationary phase and the mobile phase carries the sample through it.
Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster.
(MS) Mass Spectrometry:
Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized compounds to separate them from each other.
Compounds have distinctive fragmentation patterns that provide structural information to specifically detect compounds.
Types of Mass Spectrometers
• LC-MS (Single Quadrupole)
• LC-MS/MS (Triple Quadrupoles)
• LC-Q (Ion Traps, Linear Ion Traps)
• LC-Q-TRAPS (Quadrupole Linear Ion Traps)
• LC-TOF-MS (Time-of-Flight)
• Q-TOF-MS (Quadrupole Time-of-Flight)
• FT-MS (Fourier Transform)
LC-MS/MS (Triple Quadrupole)
The instrument can be used for quantification and identification of known biological and chemical molecules in complex extracts and mixtures.
The sum of all these instruments represents a multipurpose and efficient MS centre due to its adaptability. Versatility of the facility refers not only to Ionization modes ( ESI, APCI, APPI, ASAP and EI), but also to diversity of separation technologies available like UPC2 (SCF chromatography), UPLC, GC and Ion Chromatography. Therefore a large number of different chemical compounds can be separated and identified in this facility.
- Can be the most sensitive method for protein ID (The smaller the column the more sensitive)
- Unambiguous search results (Very few false positives)
- Only a single peptide is required for ID
- The ability to perform limited homology searches single substitution within a peptide
- Able to ID proteins from simple mixtures (~20) such as immunoprecipitates
- The data can be searched against EST databases
- Time (Both acquisition and analysis)
- Chromatography problems of NanoLC
The LC-MS/MS method development can be separated into two parts: sample extraction method development and LC-MS/MS instrument method development.
Sample Extraction Method Development
A sample extraction step may be performed using protein precipitation (PPT), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) methods. PPT is a commonly used sample extraction method, especially in the drug discovery stage. It is a fast, easy-to-apply method for small molecule analysis. The drawback of PPT is that it is not a selective method like LLE or SPE, so matrix effect or ion interference may be observed in chromatograms. LLE is the other widely used sample extraction method. It can be developed as a specific method and can generate clean sample extract solutions.
Another sample extraction method, the SPE method, separates analytes in a chromatographic way and generates a cleaner resultant solution. Extract solution prepared by LLE and SPE methods are more suitable for ultra high performance liquid chromatography (UHPLC) analysis. All three sample extraction methods can be transferred to robotic systems to increase sample preparation efficiency.
LC-MS/MS Instrument Method Development
LC-MS/MS instrument method development requires appropriate settings of LC system and MS system. For LC system setup, mobile phase, LC column, gradient, flow rate, and column temperature are the most commonly adjusted factors based on method development time, analysis run time, and expected number of samples per day.
The purpose of method development is to establish a fast, reliable method which can provide clear resolution of the analyte(s). For MS system setup, ion source parameters, mass transitions monitored in multiple reactions monitoring (MRM), and collision cell parameters are commonly-adjusted factors based on expected analyte concentration and response. The goal of adjustment is to optimize MRM transition response while remaining free of interference. The LC-MS/MS methods developed by our bioanalytical team offer expedited, sensitive, and isotopic-specific results of the analytes. LC-MS/MS method validation is necessary to obtain reliable data during new drug development. A well-developed and validated method is often used from drug development to post-marketing surveillance.