REAL DECRETO 436/2004, DE 12 DE MARZO, POR EL QUE SE ESTABLECE LA

Tandem mass spectrometryrefers to the use of two rounds of mass analysis. After an MS1 scan determines the mass of a peptide, the peptide is isolated, fragmented, and the mass spectrum of its fragments is measured. This type of scan is called anMS2 scan. MS2 spectra contain them/zand signal intensity of fragments which are often indicative of a peptide’s AA sequence.

2.4.5.1 Ion isolation

Ion isolationrefers to the accumulation of desired ions, or conversely, the filtering of undesired ions. It is used to accumulate an ion population of interest for further study. Anisolation windowis defined by itsm/z

center andm/zwidth. Ions whosem/zvalues are within the bounds of the window are isolated, and those

outside are filtered out. The amount of time spent accumulating ions is calledinjection time. An example of a quadrupole mass filter is shown in Figure 2.10. Voltages and radio frequency fields are adjusted so that only ions with the properm/zwill have stable trajectories. Ions withm/zvalues outside the bounds will not travel to the end of the quadrupole.

Typically, an isolation window is centered on either on a peptide’s monoisotopic peak or the isotopic peak with the strongest signal intensity. Narrower isolation windows centered on the desired ions have three important effects. First, advantageously, they increase the proportion of desired ions that are isolated relative to other ions because it’s less likely other ions are within the window’s bounds. Second, however, narrower windows also have decreased isolation efficiency because even ions within the window’s bounds start to have unstable trajectories if their initial velocities are outside an ever decreasing acceptable range. Longer injection times are necessary to counter the decreased isolation efficiency. Third, narrower windows will isolate a

partial isotopic distributionwhere some isotopic peaks will fall outside the isolation window. Isolation of a partial isotopic distribution leads to different isotopic distributions in subsequent mass spectra and must be accounted for during data analysis. Therefore, isolation window widths balance specificity and sensitivity, and change observed isotopic distributions.

2.4.5.2 Fragmentation

Once a population of ions is isolated, they can be fragmented in a collision cell inside the mass spectrometer (Figure 2.11). Prior to fragmentation, the ions are calledprecursors. The precursors enter the collision cell at high velocity and collide with gas particles. The collisions cause the peptides to vibrate violently until a bond is broken and two complementary B/Y fragments are created. The fragments are then

sent to a mass analyzer to measure theirm/zand signal intensity. Sequencing algorithmsdetermine the most

likely AA sequence to have generated the observed fragmentation pattern.

Fragmentation pathwaysrefer to the location and relative frequency of a break at a particular chemical bond compared to other chemical bonds. There are three bonds on each amino acid that can be broken to

Collision Cell

Collision gas

particles

Precursor

peptides ions

Fragment ions

Fragmenting

peptide

Fragment ion

Figure 2.11: Schematic of collision-induced dissociation. A collision cell is populated by gas particles such as helium, nitrogen, or argon. Peptide ions are propelled into the collision cell at high energy and collide with the gas particles causing the peptides to dissociate into smaller fragments. Some fragments will not have a charge because the protons remained associated with the complementary fragment. These neutral particles will not be affected by electromagnetic forces and therefore cannot be measured by the mass analyzer. Fragment ions, however, continue on to the next stage of the mass spectrometer.

N C C R1 O H H H N C C H OH O H R2

Amino acid residue 1 Amino acid residue 2

A ion B ion C ion X ion Y ion Z ion

Figure 2.12: Common fragmentation pathways. Two amino acids are shown connected by a peptide bond. Dashed lines indicate the three bonds that, when individually broken, will dissociate the two amino acids and provide sequence information. For notation, a break between the central carbon and the carbon of the carboxyl group can generate A and X ions. An ion that contains atoms from the peptide N-terminus to the central carbon is an A ion. An ion that contains atoms from the peptide C-terminus to the carboxyl carbon is an X ion. B ions contain atoms from the peptide N-terminus to the carboxyl carbon. Y ions contain atoms from the peptide C-terminus to the amino nitrogen. C ions contain atoms from the peptide N-terminus to the amino nitrogen. Z ions contain atoms from the peptide C-terminus to the central carbon.

P E P T I D E

B

B

B

B

B

B

Y

Y

Y

Y

Y

Y

P EPTIDE

B

Y

PE PTIDE

B

Y

PEP TIDE

B

Y

PEPT IDE

B

Y

PEPTI DE

B

Y

PEPTID E

B

Y

Figure 2.13: A peptide consisting of seven amino acids and its possible B/Y fragments are shown. The

fragment notation extends to sequences of any lengthn. The index for a fragment ion represents its amino

acid length, and therefore ranges from 1 ton₋1.

dissociate chains of amino acids from each other, and fragment ions have different notation depending on which bond was broken (Figure 2.12). Collision-induced dissociation predominantly causes breaks at peptide bonds because they are the weakest bond among the three options. This dissociation results in B and Y fragment ions. Fragments containing the amino acids from the N-terminus up to the broken peptide bond are called B ions, while fragments starting at the peptide bond and ending at the C-terminus are called Y ions. The fragments are further indexed based on the number of amino acids in the fragment (Figure 2.13). After fragmentation, the ions are sent to a mass analyzer. An example of an MS2 mass spectrum of a peptide annotated with matching B/Y fragment ions is shown in Figure 2.14. The number of pluses following an ion label indicate itscharge state: the molecule’s number of protons minus its number of electrons.