Biomolecular Mass Spectrometry: Applications to Proteins and Peptides - Molecules: Nucleation, Aggregation and Crystallization - page 65

However, there exists a simple but efficient strategy whereby very narrow
peaks for complexes can be obtained. In the work on bacterial RNA poly-
merase ( 400 kDa) this strategy has led to the discovery of a previously

undetected complex which would otherwise have been buried by the typ-
ical broad peaks of the complex (Ilag et al ., 2004). This allowed the iden-
tification of the regulatory factor Rsd as interacting with the RNA
polymerase holoenzyme and binding to sigma factor 70 displacing it
from the holoenzyme (Fig. 5). This is a strong demonstration of the power
of MS, not only in characterizing complexes but also in detecting dynamic
reorganizations that may have otherwise been overlooked. It is impor-
tant to note that such reorganizations may have important regulatory
implications.

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Ribosome

From analysis of small to medium sized complexes, MS has also tack-
led huge assemblies (MDa range) such as viruses (Bothner, Siuzdak,
2004) and the ribosome (Videler et al ., 2005). The latter is a bit more
challenging because this molecular machine is an asymmetric and het-
erogeneous complex. It is made up of three RNA chains and over 50
associated proteins. Two subunits called 50S and 30S make up the intact
70S ribosome. The crystal structure of the ribosome has been solved
(Schuwirt et al ., 2005; Wimberly et al ., 2000; Harms et al ., 2001;
Schluenzen et al ., 2000; Ban et al ., 2000). However, the stalk proteins on
the 50S subunit have rather poor electron density because they are rather
dynamic structures. Here again we see the complementary nature of MS
to X-ray crystallography because the stalk proteins seem to be the most
accessible for MS and successful measurements of intact 30S, 50S and
70S ribosomes have been made (Ilag et al ., 2005; McKay et al ., 2006;
Videler et al ., 2005). Using tandem MS on the 50S ribosomal subunit
established new heights for MS. For the first time, ions above 20000 m / z
were isolated and dissociated (Fig. 6), revealing that a population of stalk
proteins are apparently phosphorylated (Ilag et al ., 2005). This has never
been observed in E. coli ribosomes (Hanson et al ., 2003) but is a known