Effective solvation of alkaline earth ions by proline-rich proteolytic peptides of galectin-3 upon electrospray ionisation.
In an analysis of a combined chymotrypsin/AspN digest of galectin-3 by positive ion nano-electrospray ionisation mass spectrometry (nanoESI-MS) several peptides were observed which showed metal adduct ions as their most abundant ion signals. The most prominent adduct ions were observed at m/z values corresponding to [M+40]2+, [M+41]3+, and [M+42]4+ ions. Detailed investigation of the [M+40]2+ ion of the peptide GAPAGPLIVPY showed that it was not, as originally expected, a [M+H+39K]2+ adduct ion but had the composition [M+40Ca]2+. This was verified by several approaches: (i) nanoESI-MS/MS of the [M+Ca]2+ adduct ions resulted in the virtually exclusive formation of doubly charged fragment ions; (ii) mass determination by quadrupole time-of-flight (QTOF)-MS provided a preliminary identification; and (iii) accurate mass measurement using nanoESI Fourier transform ion cyclotron resonance (FTICR)-MS at a mass resolving power of 500 000 allowed the specific detection and identification of the isobaric ion pairs [M+40Ca]2+/[M+H+39K]2+ and [M+24Mg]2+/[M+H+23Na]2+. All peptides in the chymotryptic galectin-3 digest without a basic residue (K or R) showed addition of calcium as the most prominent ionisation principle. A further common feature of these nonbasic peptides was the presence of several proline residues, which is assumed to be a factor promoting the intense addition of calcium. It was observed that the common trace levels of sodium and calcium in analytical grade solvents (about 1-10 microM) are sufficient to generate the [M+H+23Na]2+ and [M+40Ca]2+ ions as the most prominent species of the peptide GAPAGPLIVPY. We conclude that the sequence motifs P-XX-P and P-XXX-P favour the solvation of alkaline earth ions in ESI-MS. In view of the successful detection of physiological Ca/protein interactions by ESI-MS, this finding may point to a solvation of Ca2+ by galectin in solution. The findings open new routes of research in the study of metal/protein and metal/peptide interactions
Lehmann
Wolf D
WD
Central Spectroscopy, German Cancer Research Center, Heidelberg, Germany. wolf.lehmann@dkfz.de
Wei
Junhua
J
Hung
Chien-Wen
CW
Gabius
Hans-Joachim
HJ
Kirsch
Dieter
D
Spengler
Bernhard
B
Kübler
Dieter
D
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry; D002918:Chymotrypsin / Q000378:metabolism; D037502:Galectin 3 / Q000737:chemistry*; D006801:Humans; D008673:Metals, Alkaline Earth / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010446:Peptide Fragments / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D037502:Galectin 3; D008673:Metals, Alkaline Earth; D010446:Peptide Fragments; D011392:Proline; D002918:Chymotrypsin; D002118:Calcium