1. MS Calibration for Protein Profiles We need calibration for –Accurate mass value Mass error: (Measured Mass – Theoretical Mass) X 10 6 ppm Theoretical Mass 100 ppm is 1Da mass error for a 10000 Da peak For linear MALDI MS, the mass error is ~100ppm –Proper binning

2. The MS generating process Time-of-flight mass spectra are recorded as intensity versus time-of-flight tables. –(m/z) =(aT+b) 2 –a and b are defined by the physical instrument dimensions and the operating parameters MS is assigned a default calibration that allows the interpretation of the spectrum. Different conditions in the instrument –Instrument errors in measuring high voltages and electrical field –Time-varying high potentials –Instrument parameters are subject to drift –Different initial kinetic energy

3. "external" calibration Applying a set of calibration constants measured in a previous experiment is known as using an "external" calibration. External calibrations are not always accurate. –experimental conditions change and may produce a systematic error in mass assignment

4. "internal" calibration This problem can be corrected using known peaks in each spectrum to calculate a calibration for that particular data set, a procedure known as using an "internal" calibration. –Internal calibrations are inherently more accurate than external calibrations; however they require at least two peaks of accurately known mass in every spectrum

5. "internal" calibration It must be pointed out that good mass assignments depend on good signals. The inconsistency of calibrant peak resolution and peak shape cause the inconsistency of internal calibration

6. Average Mass

7. Choosing Calibrant Peaks There are errors in estimating the precise centroid of the full isotopic envelope. Isotope distributions are not symmetric, so it is essential to calculate a centroid, rather than just taking the apex of the distribution. The accuracy of a centroid depends on the precise measurement of the ion current at a sufficient number of points to define the isotopic envelope precisely. Factors which can distort relative intensities across the distribution and can contribute to error in the average mass value include –overlapping distributions –low ion statistics, –detector saturation –others

11. Choosing Calibrant Peaks Consistently present in most spectra –Obtain multiple calibrant peaks Good peak resolution –Accurate mass value assignment –Smooth MS before calibration Known m/z value