Derivatization of Peptides for Analysis by High-Throughput LC-MALDI-MS/MS

 The goal of this project is to develop reagents for tagging peptides, suitable for high-throughput LC-MALDI-MS/MS (TOF/TOF).   The primary requirements of such tags are: (1) they increase signal intensity in the MS mode; and (2) they improve the quality of MS/MS (PSD and high-energy CID) spectra (i.e. more complete ion series, higher S/N) compared to unlabeled peptides. 

Early work suggested N-terminal positive charge (permanent cation) derivatization of peptides could improve their detection in MALDI and directed MS/MS fragmentation under high-energy conditions.  However, we demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI MS/MS analysis.  In contrast, N-terminal tagging with negatively-charged sulfonic acid derivatives significantly improves unimolecular fragmentation of peptides, leading to higher intensity y-ion series. The presence of a strong negative charge, however, can lead to decrease of positive MALDI MS signals for labeled peptides.

Hydrophobicity is a second factor that influences MALDI MS signals of peptides.  It is known that peptides containing aromatic amino acids, such as Phe, Tyr and Trp, usually produce higher intensity MALDI MS signals than peptides lacking aromatic residues. The reason is believed to be improved incorporation of these peptides into MALDI matrices (CHCA). 

We found that coumarin derivatives, when used as N-terminal tags, enhance intensities of MALDI MS signals of peptides. The magnitude of this effect is peptide-dependent, being the most pronounced for hydrophilic peptides and phosphopeptides (up to 50-fold enhancement in CHCA).  The enhancement factor also depends on the MALDI matrix, being the largest for 2,5-DHAP (among CHCA, 2,5-DHAP and 2,5-DHB).  For 2,5-DHB, a decrease of MALDI MS signal was observed for coumarin-labeled peptides, compared to the unlabeled peptides.  These observations further support the hypothesis that labeling peptides with coumarin tags improves their incorporation into hydrophobic MALDI matrices, such as CHCA and 2,5-DHAP.

An increase in MALDI MS signal intensity was the reason for higher ion current in the TOF/TOF MS/MS spectra of labeled peptides. On average, signal intensity in the MS/MS mode was 3 times higher for labeled peptides.  In addition, the coumarin-directed fragmentation pathway resulted in formation of b1- and a1-ions in the MS/MS spectra (these ions are not observed in MS/MS spectra of native peptides), and in many cases a higher intensity of b2- and b3-ions.  All of these factors increase the confidence scores in proteomic identification by database search, for labeled tryptic peptides compared to unlabeled digests. 

Because size, hydrophobicity and basicity of the tags are comparable with those of amino acids (e.g. tryptophan), derivatization does not impair chromatographic behavior of peptides.  This is important for the potential use of the tags for high-throughput LC-MALDI-TOF/TOF MS.   

Fig. 1.   Coumarin tags used for N-terminal derivatization of peptides (Su = N-oxysuccinimide)

Fig. 2.   MALDI MS Signal Enhancement with Pacific Blue (Coumarin tag) as a function of matrix (CHCA, DHAP and DHB)