Biomedical Engineering Reference
In-Depth Information
reduction of trypsin efficacy. As peptide mixture can be too complex for a single-
run-MS analysis, peptides can be separated by 2D HPLC chromatography, includ-
ing a strong ion exchange followed by a reverse-phase chromatography before MS
studies.
Some laboratories apply 2D-GE technique for proteomics analysis. In several
nanoparticle-binding studies, 2D-GE with subsequent mass spectrometric analysis
of the excised protein spots was used. Especially attractive is a two-dimensional
method with strong cation exchange (SCX) in one dimension and reverse-phase
HPLC separation in a second dimension, before the use of an MS/MS instrument.
Such a technique was able to resolve thousands of plasma proteins over
104 dynamic concentration ranges, without the need for depletion of abundant
proteins. Recently, a shotgun two-dimensional LC-MS/MS proteomics approach
was used to analyze plasma proteins that bind to dextran-coated iron oxide
nanoparticles [
28
].
Recently, Tenzer et al. [
29
] studied the long-lived blood plasma-derived corona on
monodispersed amorphous silica nanoparticles differing in size (20, 30, and 100 nm).
The composition of the protein corona was analyzed qualitatively and quantitatively
by liquid chromatography, mass spectrometry, one- and two-dimensional gel electro-
phoresis, and immunoblotting. 125 proteins were identified, and an enrichment of
specific lipoproteins as well as proteins involved in coagulation and the complement
pathway was observed. In contrast, immunoglobulins displayed a lower affinity for the
particles. They demonstrated that electrostatic effects alone are not the major driving
force regulating nanoparticle-protein interactions.
4.8 Fourier Transform Infrared and Raman
Spectroscopies
Raman and Fourier Transform Infrared (FTIR) spectroscopies give information
about the surface properties of NP-protein complexes and allow detecting the
protein binding onto the surface. Generally, the experimental problem associated
with vibrational spectroscopy of protein is spectral crowding. There are two main
advantages of Raman over FTIR for studying NP-protein interactions: its ability to
measure the protein-NP complexes in aqueous solution and the greater spectral
simplicity in Raman spectra than IR because the localized vibrations of double- or
triple-bond or electron-rich groups generally produce more intense Raman bands
than vibrations of single-bond or electron-poor groups. FTIR has been used to
monitor the structure of NP-bound proteins [
9
], and the protein secondary structures
are estimated based on the absorption of amide bonds (1,700-1,600 cm
1
). These
spectroscopic methods allow confirming the protein attachment onto NPs through
the appearance of additional characteristic bands.
