Figure 1.
Focusing of hydrophilic peptides by the liquid matrix, 3AQ/CHCA.
(A) Schematic representation of the on-target separation method described in this study. 3AQ/CHCA is first spotted onto the MALDI target plate, and then the proteolytic whole protein digests containing both glycosylated and nonglycosylated peptides are applied to the top surface of the liquid matrix. (B) Time course monitoring of the sample droplet on the liquid matrix. Evaporation of the sample solution allows the droplet to shrink such that the hydrophilic constituents are gradually concentrated in a focused droplet.
Figure 2.
MS spectrum of tryptic MT1-MMP digests within the central area of 3AQ/CHCA.
(A) Endogenous MT1-MMP was immunoprecipitated with anti-MT1-MMP antibody-conjugated resin from membrane lysates of wild-type HT1080 cells and was further separated by SDS-PAGE and staining with Coomassie Blue. Sialidase, which contains BSA as a carrier protein, was loaded in the light lane (denoted by asterisk). Numbers on the left of the panel represents molecular masses in kilodaltons (kDa). (B) The polypeptide band corresponding to MT1-MMP was excised, digested in-gel with trypsin. An aliquot of tryptic MT1-MMP digest derived from HT1080 cells was applied directly onto the liquid matrix 3AQ/CHCA on the MALDI target plate. MS spectrum was obtained within the central area of the liquid matrix (open circle [○]). A stereoscopic microscope image of the sample spot is shown in the left upper insert. Number represents the cumulative intensity of the top peak (arbitrary units).
Figure 3.
MS spectrum generated from glycopeptides of MT1-MMP.
An aliquot of tryptic MT1-FLAG digest was applied directly onto the liquid matrix 3AQ/CHCA. MS spectrum was obtained within the central area of the liquid matrix. The glycan moieties and the amino acid sequences of peptides including glycosylation sites of these glycopeptides were assigned by MS2 and MS3. Number represents the cumulative intensity of the top peak (arbitrary units).
Figure 4.
MS2 spectra of the ions at m/z 2,042.6, 2,245.0, 2,406.3, 2,608.6, 2,770.1, 2,972.6 and 3,134.2.
Ion peaks derived from the MS spectrum of tryptic MT1-FLAG digests at m/z 2,042.6, 2,245.0, 2,406.3, 2,608.6, 2,770.1, 2,972.6 and 3,134.2 (Fig. 3) were subjected to MS2 analysis. These fragment ions were assigned to the loss of single monosaccharides (162 and 203 Da for Hex and HexNAc, respectively) from the same peptide ion (299TTSRPSVPD307).
Figure 5.
O-glycosylation sites of MT1-MMP.
MT1-MMP is a type I transmembrane proteinase, and has a particular multidomain structure with a catalytic domain, a hinge domain, a hemopexin-like domain and a stem domain in the extracellular region. Underlines indicate glycopeptides identified in this study.
Figure 6.
Comparison of MS spectra of tryptic MT1-MMP digests containing different amounts of MT1-MMP.
(A) MT1-FLAG was serially diluted and then separated by SDS-PAGE and stained with Coomassie Blue. Sialidase, which contains BSA as a carrier protein, was loaded in the far-left lane (denoted by asterisk). Numbers on the left of the panels represent molecular masses in kilodaltons (kDa). (B) The polypeptide bands corresponding to MT1-FLAG were excised, in-gel digested with trypsin, and analyzed by MS using the liquid matrix 3AQ/CHCA. Glycopeptides contained in the whole protein digest obtained from MT1-FLAG (approximately 1.6 ng based on comparison with a BSA standard) were detected in the center of the liquid matrix. Numbers represent the cumulative intensity of the top peaks (arbitrary units). Arrowheads indicate glycopeptide ions (refer Fig. 3).