Biology Reference
In-Depth Information
3
The Breakthrough for Proteomics: Molecular Elephants in the Gas Phase
By the 1970s, mass spectrometry had become a standard analytical
method in the analysis of organic compounds. However, the appli-
cation of mass spectrometry to biological studies was hampered
due to the inexistence of suitable ionization methods for fragile
and nonvolatile biological molecules. The 1980s saw the develop-
ment of ionization techniques capable of efficiently ionizing bio-
logical molecules. This included Californium-252 plasma
desorption (
252
Cf-PD) in 1976 [
30
], fast atom bombardment
(FAB) in 1981 [
31
,
32
], matrix-assisted laser desorption/ioniza-
tion (MALDI) in 1985-1988 [
33
], and electrospray ionization
(ESI) in 1984-1988 [
34
,
35
]. FAB uses a source of neutral heavy
atoms to ionize compounds from the surface of a liquid (i.e. glyc-
erol) matrix. The breakthrough for large molecule ionization came
in 1987 when Koichi Tanaka and colleagues from Shimadzu
Corporation (Japan) were able to ionize the 34,472 Da protein
carboxypeptidase-A using what they called the “ultra fine metal
plus liquid matrix method” that combined 30 nm cobalt particles
in glycerol with a 337 nm nitrogen laser for ionization [
36
,
37
].
Simultaneously, Michael Karas and Franz Hillenkamp ionized the
67 kDa protein albumin using a nicotinic acid matrix and a 266 nm
laser [
38
]. Meanwhile, John B. Fenn and colleagues at Yale
University refined an ion source originally reported by Malcolm
Dole of Northwestern University almost two decades earlier to
develop the electrospray ionization (ESI) technique [
34
,
35
].
252
Cf-PD, FAB, ESI, and MALDI are all relatively soft ionization
technique that produce primarily protonated [M + H]
+
and depro-
tonated [M−H]
−
quasimolecular ions. However, with the develop-
ment of MALDI and ESI, the upper mass range was extended
beyond 100 kDa and this had an enormous impact on the use of
mass spectrometry in life sciences. “For the development of soft
desorption ionisation methods for mass spectrometric analyses of
biological macromolecules” John B. Fenn and Koichi Tanaka were
awarded with the Nobel Prize in Chemistry in 2002.
In ESI the liquid containing the analyte of interest is electro-
statically dispersed at the ES capillary (internal diameter <250 μm),
placed at a potential difference between +500 and +4,500 V, pro-
ducing a fine aerosol of charged droplets. In 1994, Emmett and
Caprioli [
39
] and Wilm and Mann [
40
] introduced the microelec-
trospray, later developed as nanospray [
41
], demonstrating that a
capillary flow of ~25 nL/min can sustain an electrospray at the tip
of emitters fabricated by pulling glass capillaries to a few microm-
eters. To decrease the initial droplet size, compounds that increase
the conductivity (e.g. acetic or formic acid) are customarily added
to the solution. Regardless of the size of the ESI set up, solvent
flowing at the tip of the capillary evaporates from a charged droplet
