Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 260

of a “tuning set” of a number of known organelle proteins,
against which all other proteins is compared, are averaged
along each dimension to give a vector of expected values
for each fraction,

n ), known as the cen-
troid. The distance of a given observation from this point
is a measure of the similarity of that protein's profile to the
tuning set.
2. Q uantify the similarity as a Mahalanobis distance, d M , d M =

μ = ( μ

1 ,

μ

2 ,

... , μ

( x

) T − 1 ( x is the variance-covariance

matrix of the tuning set. The Mahalanobis distance repre-
sents the distance of a given point from the center (mean)
of the “consensus proteins” in n -dimensional space, nor-
malized by the variance (and covariance) of this group of
points. Equal deviations from the centroid in two differ-
ent dimensions (fractions) may yield different contributions
to the Mahalanobis distance, depending upon the breadth
of the tuning protein distribution in those dimensions. The
metric therefore makes a measure of the similarity of a given
point to the consensus set.
3. Plot the Mahalanobis distance. Data from the double PCP-
SILAC experiments provide a distance measure for each of
the two experiments. Proteins with low values in both exper-
iments are likely organelle-associated proteins ( Fig. 15.2F ) .

− μ − μ ), where

4. Notes

1. The lysis buffer should contain protease inhibitors to pre-
vent partial digestion of the sample during the purification
procedure. However, if, e.g., lysosomes are purified and
the organelle distribution should be analyzed by enzymatic
activity measurements, protease inhibitors should be omit-
ted. Lysis buffer with detergent should be avoided when
isolating membrane enclosed organelles.
2. Discontinuous gradients are typically prepared with solu-
tions of sucrose or iodixanol by underlying solutions of
increasing density. Continuous gradients are typically pre-
pared by the use of a gradient mixer, by the centrifugation
of iodixanol solutions, or by partial diffusion of a step gradi-
ent overnight.
3. Light, medium, and heavy labeled cells refer to cells cultured
in SILAC medium with light (Lys0 and Arg0), medium
(Lys4 and Arg6), and heavy (Lys8 and Arg10) isotope-
labeled amino acids. Cells should be cultured in the SILAC
medium for at least six cell divisions to fully incorporate
the SILAC amino acids. Incomplete labeling might result