LC-MS data acquisition and pre-processing

Jessica Cooperstone

Chromatography

  • The process of separating components of a mixture
  • Based on an analytes’ difference in partitioning between a stationary and mobile phases

https://en.wikipedia.org/wiki/Chromatography

Types of liquid chromatography

  • Reversed phase
  • Normal phase
  • HILIC (hydrophilic interaction liquid chromatography)

Mass spectrometry

Specifics about MS

  • Requires gas phase ionization (no charge, no signal!)

  • Different ionization methods (e.g., ESI vs. APCI)

  • Inherently non-quantitative

    • A higher signal for compound A than B doesn’t necessarily mean compound A is present in higher concentration

  • Ion suppression in complex mixtures

Mass analyzer: time of flight (TOF or QTOF)

Allen and McWhinney, Clin Biochem Red 2019

Mass analyzer: Orbitrap

https://www.youtube.com/watch?v=j2_FVJr9xNk

MS output

MS data is really 3D

Different ways to express a mass

  • Nominal mass: sum of the integer masses of the constituent elements of a molecule (C=12, H=1)

  • Monoisotopic mass: mass of a molecular given empirical formulate calculated using the exact mass of the most abundance isotope of each element (C=12.0000, H=1.0078)

  • Average mass: mass of a molecule given empirical formulate calculated using the weighted average mass for each element by isotopic abundance (C=12.0112, H=1.00797)

  • C20H42

    • Nominal mass: (20 × 12) + (42 × 1) = 282

    • Monoisotopic mass: (20 × 12.0000) + (42 × 1.0078) = 282.3276

    • Average mass: (20 × 12.0112) + (42 × 1.00797) = 282.5587

Try calculating the monoisotopic mass

Tomatidine C27H45NO2

Example: Rutin

Compare experimental to exact mass in working towards a compound ID

  • Exact mass is calculated (theoretical)
  • Accurate mass is experimental determined (observed)

Example:

  • rutin observed (positive mode): 611.1613 m/z
  • rutin theoretical: 610.1534

Why you need high mass accuracy for metabolomics

Standards for identity reporting

Fig. adapted from Sumner et al., Metabolomics 2007

Converting raw spectra into a feature table (i.e., pre-processing)

  • Vendor software:

    • Agilent: Profinder

    • Waters: Progenesis

    • Thermo: Compound Discoverer

    • Bruker: MetaboScape

    • Shimadzu: LabSolutions

  • Open source software:

Castillo et al., Chemometrics Intelligent Lab Systems 2011

© Jessica Cooperstone, 2024