Important considerations for obtaining high quality signal are sample solubility, matrix selection, ionization characteristics, salt content and purity. Factors affecting data interpretation include quantitation, molecular weight calculation, isotope patterns, calibration/accuracy, sensitivity, and speed.

Quantitation

Do the ion intensities correlate to the relative amounts of each component? MALDI does not provide quantitative information unless the compound has been calibrated against an internal standard. ESI provides some quantitative information based on external calibration but internal calibration is more accurate. Following are some factors that affect the ability of a mass spectrometer to perform quantitative measurements:

• Averaging: More averaging results in fewer errors associated with random noise
• Quantity of material: common sense
• Dynamic signal range: ion traps have small dynamic ranges. Quadrupoles and TOFs have much larger range.
• Ionization technique: ESI’s stable signal provides better quantitation then MALDI.
• Compound’s functional groups: Functional groups on a molecule can drastically affect the ionization properties. For instance, an amine will pick up a proton far more efficiently than an amide. Therefore in order to obtain good quantitative data an internal standard with comparable ionization characteristics is desirable.
• Choice of internal standards: An internal standard with comparable ionization characteristics to the compound of interest allows for consistent relative signal stability. The best choice is an isotopically labeled internal standard.
• Consistent sample handling: common sense

Ability of a molecule to become ionized is closely related to its functional groups. The best quantitation is obtained when a compound is calibrated against an internal standard similar to the molecule in question.

Calculating Molecular Weight

There are 3 different ways to calculate mass from molecular mass formula. Each is used for a specific reason.

1. Monoisotopic Mass: The mass of an ion is calculated using the exact mass of the most abundant isotope. Monoisotopic mass is used when the individual are distinguishable.
2. Average Mass: The mass of an ion calculated using average atomic weight of all the isotopes. Used when individual masses are not distinguishable.
3. Nominal Mass: The mass of an ion is calculated using integer mass of the most abundant isotope. e.g. C13, N14, etc. Nominal mass is not used very often.

The resolving power of a mass spectrometer is very important in calculating mass of a molecule.

Isotope Patterns

Isotope patterns can be a great source of information. The spacing of isotopes indicate the charge state. ½ spacing = 2+ charge state, 1/3 spacing = 3+ charge state. Certain elements have distinct isotope patterns which help in identifying them.

Solubility

Sample solubility is absolutely critical in obtaining quality data. The solvent of matrix allows the sample to be transformed to gas phase, thus playing a critical role in ionization.

Timing

Analyzing the sample ASAP after it has been prepared is important, as it is quite common for compounds to decompose or even react with the solvent in a relatively short time. A problem associated with hydrophobic compound is loss of the sample to the container’s surface.

Calibration/Accuracy

Protein identification depends on accuracy of mass measurements. For high accuracy, it is often necessary to have an internal standard present or at least some reference compound. Standard compounds are used to calibrate a mass spectrometer’s mass analyzer with respect to how it measure m/z. Calibration is generally performed using a standard mixture, such as PPG, that generates a reliable source of known ions that cover the mass range of interest.

It is important to distinguish between internal and external calibration. External calibration refers to the instrument being calibrated followed by analysis without the present of a calibrant. Internal calibration refers to analyses that are performed with a calibrant present to improve accuracy.

Sample purity

Sample purity maximizes sensitivity. MALDI samples can be cleaned and desalted with ZipTip or a droplet of cold water. Dialysis for ESI.

Sensitivity/Saturation

If too little sample is used, the instrument will be unable to detect a signal. Too much sample can skew the intensity profile of the ions. Higher concentrations can also amplify the effects of impurities. It is important to be within the correct range of your instrument.

Ionization characteristics

The types of functional groups on a molecule will often determine how a compound should be analyzed.