Protein Identification

Introduction to Mass Spectrometry

The common procedure for identification by mass spectrometry include (1) 2D-PAGE or other factionation procedure, (2) digestion with trypsin which are then (3) fed into either a MALDI-MS or a MS/MS or ESI-MS and (4) database comparison using algorithms.

For proteomics, one wants good data on peptide masses (MALDI-TOF MS) or good data that describes peptide fragmentation (ESI tandem MS). This requires sensitivity (amount of proteins capable of being detected), resolution (how well one can distinguish ions of very similar m/z values; usually need to be able to distinguish ions differing in m/z values of at least one Dalton (i.e., the mass of a single H atoms) and mass accuracy (as close as possible to the real value).

A mass spectrometer is an instrument employed to produce ions from atoms or molecules (the source) which are then separated according to their charge-to-mass (m/z) ratios and detected. In peptide mass fingerprinting, MS is used to measure the masses of proteolytic peptide fragments. The protein is then identified by matching the measured peptide masses to corresponding petpide masses form protein databases.

The way this all works is that trypitc digestion of each protein yeild a specific number of peptides of specific mass. The peptides then are analyzed by MS to obtain m/z values, which can be converted to masses. For example, tyrptic digestion of human hemoglobin alpha chain yeild 14 tryptic peptides, of which one petide has the mass of 1528.7348 Da. Thus, the singly charged ion of this peptide has an m/z value of 1529.7348. If wer were able to measure only to the nearest whole m/z value (i.e., 1529), there would be 478 matches. But the more accurate the m/z measurement provided, the fewer choices we would get. Accurate protein identifications usually also require multiple peptide matches. Even the best possible mass match would be unable to tell you whether the peptide you analyzed was from human or mouse hemoglobin. However, any real tryptic digestion would yeild multiple peptides and given you multiple m/z values to search against a database. (such as SWISS-PROT), OWL and NCBInr)

Here is another example: Suppose your peptide is AVAGCAGAR. Each residue in this has an amide NH group at one end, a C=O group at the other, and an alpha carbon with one proton in the iddle. The side chains give each amino acid its special chemistry are attached to the alpha carbon. Each amino acid has an average residue mass. For example, G has an average residue mass of 57.05. We would need to take on an extra proton (1 amu) to the N terminal residue and an extra OH (17 amu) to the C terminal. The cumulative nubmer of masses of each residue is how the MS sees this peptide.

When peptide ions collide with neutral gas atoms in the collision cell of a triple quad or ion trap (below), the kinetic energy they absorb induces fragmentation. In the most commonly observed cleavage, the bond between the carbonyl O and the amide N is cleave to form a "y-ion" and a "b-ion". A y-ion is a fragment in which the + charge is retained on the C terminus of the original peptide ion and a b-ion is a fragment in which the charge is retained on the N terminal portion of the original peptide ion. Doubly charged ions are most likely to have charges at the opposite ends of the molecule. When these peptide ions fragment, both a b-ion and y-ion are formed. When singly charge ions fragment, either a b-ion or a y-ion is formed. The other half of the peptide is lost as a neutral fragment. One gets twice as much information from fragmentation of doubly charged as opposed to singly charged ions.

Lets take an example of glycine (C2H5O2N). As an uncharged residue, it has a mass of 75, as an ion (GlyH+) 76, as a fragemtn 57, as a b1ion 58, as a y1ion 76.

1. Ionization methods

Matrix-Assisted Laser Desorption (MALDI):

In a MALDI, 1) the sample to be analyzed is mixed with a chemical matrix which typically contains a small organic molecule with a desirable chromophore that absorbs light at a specific wavelenght, 2) the admixture of sample and matrix is then spotted onto a small plate/slide, 3) the target is placed into the source equipped with a laser which fires a beam of light at the target. The matrix chemicals absorb photons from the beam and become electronically excited. This energy is then transferred to the peptides or proteins in the same which are then ejected from the target surface into the gas phase.

This ionization process produces both positive and negative ions, depending on the nature of the sample. For peptides and proteins, the positive ions are usually the species of interest. The positive ions are formed by accepting a proton as they are ejected form the matrix. Each peptide molecule tends to pick up a single proton. For a peptide of say mass 1032, the addition of a proton and its one positive charge makes the m/z value 1003 for the [M+H]+ ion.

Electrospray ionization (ESI): In an ESI the sample enters the source through a flow stream (often from the HPLC) and passes through a needle held at high voltage. As the flow stream exits the needle, it sprays out in a fine mist of droplets. In contrast to MALDI, in which the sample is a dried, crystalline admixture of peptide sample and matrix, the peptides or proteins to be analyzed by ESI are in aqueous solution. Peptides exist as ions in solution because they contain functional groups whose ionization is controlled by the pH of the solution. Thus, carboxylic acids are protonated (unionized) below pH 3.0 and ionized at pH values above about 5.0. In contrast, N-terminal amines and histidine nitrogens are weak bases that are ionized below pH 7.0. The nitrogen functional groups of lysine and arginine are usually ionized below pH 8.5. This means that at acidic pH values (i.e., pH 3.5 and below), protonation of the amines will confer overall net positive charge to peptides and proteins. At basic pH, deprotonation of the amines and carboxyl groups confers a more negative overall charge.

A unique characteristic of ESI is the production of multiply charge ions from proteins and peptides. Many peptides bear multiple proton accepting sites and can exist as singly charge or multiply charged ions in solution. This is particularly true of peptides derived by tryptic digestion, as they bear lysine or arginine residues at their C termini as well as N-terminal amino groups, both of which may be protonated in acidic solution. Multiple charging serves the added purpose of forming ions that are within the mass range of quadrupole and ion-trap mass analyzers (below) which have more limited mass range than the TOF analyzers. For example, the absolute mass of a singly protonated 20 kDa protein (m/z=20,001) is well outside the mass range of a quadrupole mass analyzer which typically extends to 32 kDa of sometimes 4kDa. However, the typical 20 kDa protein will accept anywhere form 10-30 protons in solution. Thus for a population of these protein molecules in solution, some will have 20 protons and a m/z of 20,020/20=1001, some will have 21 protons and a m/z of 20,0021/21=953, and so on.

Three types of tandem mass analyzers are commonly paired with ESI sources. These are the triple quadrupole, the ion trap and the TOF discussed below. Those analyzers all perform the same type of analysis. From a mixture of peptide ions generated by the ESI source, the analyzers select a single m/z species. This ion is then subjected to collision induced dissociation which induces fragmentations of the peptide into fragment ions and neutral fragments. The fragment ions are then analyzed on the basis of their m/z to produce a product ion spectrum. which permits the sequence of the peptide to be deduced. The location of peptide modification also can be established.

2. Ion Separation

Triple Quadrupole: consists of 4 meal rods arranged in aprallel. Direct current and radiofrequency voltages are applied to the rods. Depending on the voltage applied, ions of a specific m/z value will pass through the quadrupole, whereas ions of greater or less m/z values will fly outwards an fail to pass through the quadrupole.

Ion Trap: Whereas triple quads analyze and peptide ions as they pass through the analyzer, ion traps collect and store ions in order to perform analyses on them. Ions collected in the trap are maintained in orbits wby a combination of DC and radiofrequency voltages. A particular ion of interest is selected and the trap voltages are adjusted to eject ions of all other m/z values. The voltages of the trap then are quickly increased to increase the energies of the remaining ions, which results in energetic collisions of the peptide ions with the helium gas atoms in the trap and induces fragmentation of the ions which are then caught in the trap and scanned out according to their m/z values.

Time of Flight (TOF); The ions formed in a MALDI source are extraced and directed into this analyzer. TOF measures the time it takes for the ions to fly from one end of the analyzer to the other and strike the detector. The speed with which the ions move is proportional to their m/z values. The greater the m/z, the faster they fly.

Having ions simply move from start to finish in a linear mode did not produce a good resolution (ability to distinguish ions of slightly different m/z values). This problem was solved with a reflectron

Ion Detection

Typically detection using a conversion dynode which converts the ions into an electrical signal.

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