Summary of Methods Used in the Study of Pesticide Biotransformation and Disposition - Pesticide Biotransformation and Disposition - page 20

Fractionation (e.g., FPLC; FFE)

Extraction

Cells

Sample
(e.g., bacteria)

Harvest
Wash
Precipitation
Isolation, etc.

Proteins

Centrifugation

Extraction

Top-down Bottom-up

MudPIT
Gel-free Gel-base

DNA/RNA

Supernatant

Separation

Digestion

DGGE
PRC
RT-PRC
Sequencing
Cloning
Expression

2D-GE DIGE 1D-GE

Ion exchange
column

Extraction Filtration

Image analysis

Digestion

Acidic/Neutral

Digestion

MALDI TOF
FTMS
Orbitrap MS

Derivatization

MALDI TOF LC/IT MS LC/IT MS

LC/MS
(e.g., QQQ, Q-TOF)

GC/MS

Database search and amino acid sequence

Figure 2.2 Schematic of “multiomics” approaches applied in metabolism studies of pesticides, in
which MS-based proteomics and metabolomics workflow is emphasized. Metabolites in the super-
natant of bacterial samples can be analyzed by gas chromatography/mass spectrometry (GC/MS) and
liquid chromatography/mass spectrometry (LC/MS). DNA and RNA extracted from cells can be ana-
lyzed by denatured gradient gel electrophoresis (DGGE), polymerase chain reaction (PCR), real-time
PCR (RT-PCR), sequencing, cloning, and other molecular techniques. Proteins extracted from bacterial
cells can be analyzed via top-down or bottom-up approaches. In the bottom-up approach, a mix-
ture of proteins can be separated by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and
then analyzed by MS. Alternatively, the protein mixture can be directly digested into a collection of
peptides that are then separated and determined by multidimensional chromatography online cou-
pled to tandem mass spectrometric analyses [i.e., multidimensional protein identification technology
(MudPIT)]. In the top-down approach, intact proteins are fractionated into less complex protein mix-
tures for MS analysis. Bioinformatics including database search and amino acid sequence alignment
is then conducted to identify and characterize the proteins and peptides. DIGE, difference gel elec-
trophoresis; FFE, free-flow electrophoresis; FPLC, fast protein liquid chromatography; FTMS, Fourier
transform mass spectrometry; MALDI TOF, matrix-assisted laser desorption ionization time-of-flight;
QQQ, triple quadrupole; Q-TOF, quadrupole time-of-flight. (Adapted from Hayes and Krieger, 2010.)

separated by isoelectric point. Recent improvements in 2D-GE techniques include the
development of immobilized pH gradients, very sensitive staining methods, and more
powerful image analysis systems. A related technique, differential in-gel electrophoresis,
has improved the reproducibility of 2D-GE by minimizing variations between gels.
These methods are commonly combined with MALDI-TOF MS (as discussed under
Analysis).