Why does MALDI analysis of my oligos containing one or more Fluorescein-dTs give an incorrect mass even though they give only a single, fluorescent band on a PAGE gel?

This is an artifact of the MALDI analysis. The lasers that are used to ablate the MALDI matrix are generally between 308 and 355 nm, with the most frequently used laser line at 337 nm. When a laser hits a strong absorbance band of a molecule, often photochemistry starts to occur - which often leads to cleavage reactions. The Fluorescein-dT has strong UV absorbance in this region which appears that a 135 m/z fragment is being blown off from the molecule in a rather consistent manner. For instance, for your oligos D1 and D2, which have 5 and 3 Flu-dTs, respectively, the observed mass difference is -676 Da and -406 Da, which nicely fits with the loss of a 135 mw fragment: 5 x 135 (675) and 3 x 135 (405). We haven't seen any papers that identify this 135 m/z fragment, but it's clear to us that that is what's occurring. Changing the matrix used in the procedure may help. Matrix 2,4,6-trihydroxyacetophenone has been used successfully to analyze a fluorescein-labeled oligos, though the safest bet is to use Electrospray MS rather than MALDI for mass spec analysis.