Current NMR Strategies for Biomarker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 88

FIGURE 1 1 H NMR Spectra (500 MHz) recorded for second-trimester urine and plasma of a healthy pregnant woman: (A)
urine, standard 1D spectrum; (B) blood plasma, standard 1D (top), T 2 T2-edited (middle), and diffusion-edited (bottom) spectra.
Legend: (1) b -hydroxybutyrate, (2) 3-aminoisobutyrate, (3) lactate, (4) threonine, (5) alanine, (6) g -aminobutyrate (GABA), (7)
succinate, (8) citrate, (9) dimethylamine, (10) creatine, (11) creatinine, (12) trimethylamine N-oxide (TMAO), (13) betaine, (14)
glycine, (15) guadinoacetate, (16) trigonelline, (17) glucose, (18) histidine, (19) phenylacetylglycine, (20) hippurate, (21)
formate, (22) N-methyl-nicotinamide, (23) lipid CH 3 , (24) lipid (CH 2 ) n , (25) glycoproteins, (26) choline, (27) lipid C H

¼ CH, (28)

valine, (29) glutamine, (30) tyrosine, (31) lipid C H 2 CH 2 CO, (32) lipid C H 2 CH ¼ CH, (33) lipid CH ¼ CHC H 2 CH ¼ CH, (34)

albumin lysyl ε -CH 2 . (Adapted with permission from reference #33. Copyright 2011 American Chemical Society.)

in urine lowers NMR sensitivity. All efforts to
minimize the addition of extraneous salt should
be made.

contain catalytically active proteins such as
proteases; therefore, it is advantageous to phys-
ically remove the protein content, especially for
longer NMR data acquisition. Addition of
anything that can contribute extra NMR
signals, such as protease inhibitor cocktails
routinely used in proteomics

Biological Fluids with Macromolecules
The NMR spectra of biological fluids con-

taining macromolecules (i.e., serum and
plasma) are dominated by signals from proteins
and lipoproteins ( Figure 1 b), which appear as
very broad overlapping signals. When metabo-
lites are bound to proteins, their NMR peaks
are broadened. For low af

studies, is

avoided. Macromolecules are physically

removed by filtration through a 3 kDa molec-

ular weight cutoff filter or precipitated with

organic solvents or acids. The NMR signals
from macromolecules have shorter transverse
relaxation rates compared to those from metab-
olites and this can be exploited to remove their
contribution spectroscopically, as discussed in
more detail later in this chapter.
Currently, there exists no single chemical
extraction technique that will quantitatively

nity protein binding
metabolites, protein removal will remove this
broadening. Reducing the viscosity of the solu-
tion also sharpens peaks. Stolzenburg et al. rec-
ommended dilution with a buffer (0.28 M, pH
7.4) instead of 0.9% saline. 37 Serum and plasma