Analysis of Proteins Using DIGE and MALDI Mass Spectrometry - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 54

Fig. 1. 1D gel of decreasing amounts of NHS-Cy5-labeled BDNF. Amounts loaded on the gel
range from 1 μ g to 0.1 ng, as indicated at the bottom of the gel ( a ). The molecular weights
of the protein standards are as indicated. Only the fi rst three amounts are visible when the
imager parameters are set below saturation values; ( b ) protein amounts in the 0.1 ng range
can be visualized by increasing the imager parameters (PMT Voltage) above saturation values
(>600 V). See text for details.

and letting the NHS dyes label the proteins further than 5%.
However, the experiment is presented based on the total protein
amount loaded and not on the extension of labeling.
Brain-derived neurotrophic factor (BDNF; accession number
CAA62632; calculated molecular weight of 27,741 g/mol) was
labeled with Cy dyes following labeling and cleanup procedures as
described elsewhere (( 1 ) and Chapter 10

). A dilution series of this
protein was run on a one-dimensional (1D) SDS PAGE gel (see
Fig. 1 ). The amounts of protein loaded on the gel ranged from
1 g (36.0 pmol) to 0.1 ng (3.6 fmol) as indicated in Fig. 1a .

The gel was visualized with the Typhoon TRIO+ (GE Healthcare)
scanner using the photomultiplier tube (PMT) setting below
saturation levels. Protein amounts 1

μ

g and 100 and 50 ng were
visible using these visualization parameters; by increasing PMT
voltages above saturation levels (usually >600 V), it is possible to
visualize lower amounts (down to 0.1 ng) as indicated in Fig. 1b .
All protein bands indicated in Fig. 1a were digested and subjected
to MALDI-MS analysis as described above. A variable number of
BDNF peptides were identifi ed (see Table 1 ), with a greater num-
ber of peptides identifi ed in the more concentrated samples. Protein
amounts 1

μ

g and 100, 50, and 20 ng were identifi ed correctly as
BDNF; however, lower amounts of protein were not identifi ed.
These results suggest that although it is possible to visualize and
analyze protein changes in the low femtomole range (3.6 fmol in
this example) with 2D DIGE, MALDI MS, as described here, is
reliable for protein identifi cation only in the 1-40 pmol range.

μ

To determine whether or not the presence of the Cy dyes affected
the MS-based protein identifi cation process, we run two 2D gels,
both gels containing a total of 20

3.5.2. MS Identifi cation
of Cy-Labeled vs.
Nonlabeled Proteins μ g of a protein standard mixture.