OTHER INSTRUMENTAL METHODS 151
trometer, followed by fragmentation of specifi c sample ions inside a second
spectrometer. The process results in identifi cation of the fragment ions with
the goal of identifying one large protein by its primary sequence or compo-
nents of a complex protein mixture. MS – MS also enables specifi c compounds
to be detected in complex mixtures because of their specifi c and characteristic
fragmentation patterns. Usually, a tandem mass spectrometer will contain
two analyzers, with the following common combinations: (1) quadrupole –
quadrupole; (2) magnetic sector – quadrupole; (3) magnetic sector – magnetic
sector; and (4) quadrupole – time - of - fl ight (TOF). In the peptide sequencing
experiment, the fi rst analyzer is used to select user - specifi ed sample ions arising
from a particular component, usually the (M + H) + ions. The chosen ions pass
into a collision cell where they are bombarded by gas molecules that cause
the fragment ions to form. The process is known as collision - induced dissocia-
tion (CID). The second analyzer then separates the fragment ions according
to theirm / z ratios. Polypeptides may fragment along their side chains or along
the polypeptide backbone. High - energy collisions, such as in a magnetic
sector – magnetic sector MS – MS, will result in many different types of side -
chain cleavage. Quadrupole – quadrupole or quadrupole – TOF mass spectrom-
eters generate low - energy fragmentations with fewer side - chain fragmentations.
Polypeptide backbone bonds may fragment at the backbone NH – CH, CH –
CO, or CO – NH bonds, resulting in ions called a,b, or c if they have the charge
retained on the N - terminal fragment or the x , y , or z ions if they have the
charge retained on the C - terminal fragment. The most common cleavage site
occurs at the CO – NH bonds, giving rise to “ b ” or “ y ” ions. Fragments contain-
ing more than one amino acid residue will be identifi ed by subscripts; that is,
b 2 is an N - terminal fragment with two aa residues, and y 4 is a C - terminal frag-
ment of four aa residues. The mass difference between two adjacent “ b ” ions
or “ y ” ions is indicative of a particular amino acid residue. Additionally, the
so - called immonium ion is produced for many amino acid residues in frag-
menting peptides, and these often have distinctive mass spectrum signatures
as well (see Figure 3.29 ).
Tables have been produced that indicate the most common molecular
masses found for amino acid residues in mass spectrometric analyses.
A protein identifi cation study might proceed in the following manner. First,
the protein is analyzed by mass spectrometry to determine its molecular mass
to within 0.01%. Second, the protein is digested with an enzyme, commonly
trypsin. The enzyme trypsin cleaves polypeptide chains at points following
lysine and arginine residues. Using this proteolytic enzyme ensures that each
Figure 3.29 The Immonium Ion.H 2 N+ CRH