Urinary Proteome Profiling Using 2D-DIGE and LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 291

Urine is an easily and non-invasively obtained bio-fluid that
can potentially act as a source of biomarkers. This possibility has
been explored by several studies ( 4 , 5 ) ; indeed more than 1500
proteins have recently been identified in the urine of healthy
donors ( 6 ) and this number is likely to increase. Almost half of all
urinary proteins are soluble and are the product of the glomeru-
lar filtration of plasma ( 7 ) , a substantial proportion of which arise
from extrarenal sources ( 8 ) . Large anionic proteins such as bile
salt-dependent lipase, a 110 kDa pancreatic protein, have also
been detected in urine ( 9 ) , suggesting the 'leakage' of diverse
proteins into urine. In addition, the urine protein profile is less
complex when compared to plasma, its proteins are thermostable
( 10 ) and albumin and uromodulin comprise a lesser proportion of
the urinary proteome. Therefore sample processing requires less
pre-cleaning/fractionation.
The recent rapid evolution of new technologies has led to
a variety of proteomic techniques being available to interrogate
the proteome on a large-scale permitting simultaneous study of
numerous proteins from multiple biological samples. Broadly
speaking these are divided into gel-based and non-gel-based
techniques. Gel-based proteomics (2D polyacrylamide gel elec-
trophoresis, 2D-PAGE) has become extremely popular since first
described in 1975 by O'Farrell ( 11 ) . The development of 2D
difference in gel electrophoresis using positively charged, amine
reactive and molecular weight-matched fluorescent cyanine dyes
(Cy2, Cy3 and Cy5) (2D-DIGE) significantly improved accuracy
and led to more precise quantitation over a wider dynamic range
( 12 ) . The increased dynamic detection range increased the sen-
sitivity of the technique with the added advantages of reduced
inter-gel variability, number of gels required, accurate spot match-
ing and compatibility with the identification of protein spots using
mass spectrometry (MS) ( 13 , 14 ) .
Gel-free strategies such as ICAT (isotope-coded affinity tag-
ging), iTRAQ (isobaric tags for relative and absolute quantita-
tion) and ESI MS/MS (electron spray ionisation tandem mass
spectrometry) rely on liquid chromatography (LC) for protein
separation interfaced with high-end mass spectrometers for pro-
tein identification ( 15 ) . Such techniques can be automated and
have a reduced sample requirement and often identify different
subsets of regulated proteins making them complementary to
gel-based techniques ( 15 ) . Developments at the LC interface by
a number of leading manufacturers have also greatly increased
the discovery range and sensitivity available to research groups
in the proteomic field. In fact LC and mass spectrometry (LC-
MS/MS) have become central to protein identification and quan-
tification, and many LC-MS/MS workflows have been developed
and applied to proteomics research.