ABRF Peptide Sequencing by Mass Spectrometry Quiz (2000)

Introduction

Prior to its demise, the ABRF Mass Spectrometry Committee prepared four “tryptic” peptides whose sequences did not match anything in the public nucleotide or protein sequence databases. For two of these peptides, data was acquired using an ion trap mass spectrometer, and for the other two a quadrupole / time-of-flight hybrid mass spectrometer (Qtof) was used. Using these data, a MS/MS peptide sequencing quiz was prepared for the ABRF membership, where the aim was four-fold. First, this was a sort of late final exam for those who took the 1999 ABRF course taught by Prof. Don Hunt. Second, the results might indicate the need for similar additional ABRF courses. Third, the anonymous results allowed individuals to assess their own level of proficiency at this task. Fourth, it allowed a comparison of the relative merits of two types of mass spectrometers most commonly used for peptide sequencing.

This study is closed, and the results and answers can be found here. I suppose one could still take the quiz, but you’ll have to grade yourself.

The Quiz

Check out the sequencing tutorial, if you need instructions on sequencing tryptic peptides using low energy CID data.

Ion trap data

Data for two peptides was acquired, where the first one is the easiest. To aid in the verification of your proposed sequences, MS3 spectra were acquired for both peptides. The following are all standard gif files:

Qtof data

The next two problems were obtained from a quadrupole / time-of-flight hybrid mass spectrometer (Qtof), where the third peptide is easier than the fourth. The fourth peptide was also subjected to trypsin in the presence of a 1:1 mixture of 16O and 18O water, which labeled the C-terminus with a mixture of the two stable isotopes. The precursor resolution was set wide enough to pass both the 16O and 18O labeled peptide, such that C-terminal fragment ions in the MS/MS spectrum exhibited two mass unit doublets. There are two spectra shown, where the raw data is on top, and the spectrum beneath it is the same data subjected to a y-ion filter.