150 INSTRUMENTAL METHODS
mentioned here may be interfaced with ESI, whereas MALDI is not usually
coupled to a quadrupole analyzer. More term defi nitions can be found at
http://www.ibiblio.org/pub/academic/chemistry/iupac/Download/publications
/analytical_compendium/Cha12sec2.pdf. The mass spectrometer ’ s detector
monitors ion current, amplifi es it, then transmits it to the data system where
it is recorded as a mass spectrum. Normally, the m / z values are plotted against
their intensities, showing the number of components in the sample, the molec-
ular mass of each component and its relative abundance. The common detec-
tors are the photomultiplier, the electron multiplier, and the micro - channel
plate.
The m / z ratio for biomolecules, as they ionize and pass through the mass
spectrometer ’ s analyzer, depends on a number of factors. For instance, using
ESI, samples with molecular masses (M) up to ∼ 1.2 kDa produce singly charged
molecular ions, either (M + H) + in positive ionization mode or (M − H) − in nega-
tive ionization mode. In the singly charged case, the m / z value corresponds to
the molecular mass. Proteins and peptides analyze better in positive ionization
mode (facilitated by addition of a small amount of acid, usually formic acid),
whereas oligonucleotides analyze better in negative ionization mode. In positive
ionization mode, (M + Na) + , (M + K) + , and (M + NH 4 ) + ions may also be pro-
duced in which cases the molecular mass is higher than expected by 23, 39, and
18 atomic mass units, amu, respectively. A common negative ionization mode
ion includes that having the chloride ion, Cl − , +35 amu. Samples having molecu-
lar weights greater than∼ 1.2 kDa give rise to multiply charged molecular -
related ions such as (M + n H) n + in positive ionization mode and (M − n H)n− i n
negative ionization mode. The mass - to - charge ratio, m / z , will be expressed as
mz
n
n/
()
=
MW+ H+
(3.47)
where m / z is the mass - to - charge ratio marked on the mass spectrum ’ s abscissa,
MW is the molecular mass of the sample, n is the integer charges on the ions,
and H is amu of a proton, 1.008 Da.
Usually the number of charges on an ion will not be known, but it can be
calculated using a formula based on two different ions appearing in the spec-
trum. Actually, the molecular mass of a sample can be calculated automatically,
or semiautomatically, by the processing software associated with the mass
spectrometer. Experimentally, the automatic calculation of molecular mass is
very helpful because a complex peptide or protein mixture will display anm / z
spectrum with several overlapping series of multiply charged ions.
Peptide and protein sequencing and its importance in the proteomics fi eld
were discussed in Section 2.2.3. The following gives a brief description of the
mass spectrometry methods used to achieve sequencing. First, to produce
protein or oligonucleotide structural/sequence information by mass spectro-
metric techniques, one needs to use tandem mass spectrometry (MS – MS). In
this technique, a sample is fi rst fragmented and analyzed in one mass spec-