148 INSTRUMENTAL METHODS
docking sites of 10% could be detected by time - resolved X - ray diffraction.
The GIF - formatted movie illustrating the CO photodissociation and move-
ment to the proximal pocket is available from a link in the HTML online
version of thisBiochemistry 2001 article.
Schotte and co - workers demonstrated 150 - ps time - resolved X - ray crystal-
lography of the MbCO mutant system (leu29 replaced by phe, L29F) in 2003.^55
Their technique characterized the L29F myoglobin structure as it evolved
from the carboxy to the deoxy state. One can access a QuickTime ™ version
of the movie from the supplemental material accompanying the onlineScience
article. The movie is described in the supplemental materials in the following
manner. A moving average of the time - resolved electron density maps was
constructed from the image with laser off at time points: 100 ps, 316 ps, 1 ns,
3.16 ns, 31.6 ns, 316 ns, and 3.16 μ s (each frame represents a weighted average
of the electron density from adjacent time points). The numerical time indica-
tor advances at the midpoint between adjacent time points. The yellow circles
enclose electron density likely arising from CO; the solid lines switch to dashed
lines when CO departs from that site. Figure 4 of reference 55 contains an
informative legend that decodes what is happening during the movie. The
reference 55 authors believe that this work extends the time resolution of
experimental crystallography into the same time domain available in molecu-
lar dynamics simulations (see Section 4.3.2 ). In the future, they believe that
X - ray free electron lasers, now under development, will deliver intense X - ray
pulses shorter than 100 fs, allowing further experimental study of dynamic
enzyme systems. More recent experimental results for MbCO systems studied
by time - resolved crystallography can be found in reference 56.
3.7.3 Mass Spectrometry,
Mass spectrometry (MS) is probably a familiar tool to chemistry and biology
students as a technique commonly used to measure the molecular mass of a
sample. Often, MS is used in tandem with other techniques for chromatic
separation of the sample before mass measurement. Some common hyphen-
ated techniques include: HPLC - MS, high - pressure liquid chromatography
coupled to MS; GC - MS, gas chromatography coupled to MS; or CE - MS, capil-
lary electrophoresis coupled to MS.
For large molecules such as biomolecules, molecular masses can usually be
measured to an accuracy of 0.01% of the total molecular mass — that is, within
4 Da for a sample of 40 kDa. Structural information on biomolecules can be
gained using tandem mass spectrometers — the MS – MS techniques. One frag-
ments the sample inside the instrument and analyzes the products generated
in the second mass spectrometer. This technique is used for protein, peptide,
and oligonucleotide sequencing, as will be described in more detail below.
Mass spectrometry studies on proteins can determine the purity of the
sample, verify amino acid substitutions in mutants, detect post - translational
modifi cations, or calculate the number of disulfi de bridges. Amino acid