Autoflex 3 spectrometer

A triple variant of human K2_hPg (C⁴G/E⁵⁶ D/L⁷²Y) that displays ~5-fold enhanced affinity. without loss of selectivity for PAM, as well as the inability to aggregate through disulfide bonding, compared to WT-K2_hPg, was expressed in Pichia pastoris GS115 cells as described in detail previously (Nilsen et al., 1997 ; Wang et al., 2010a (link); Wang et al., 2010b (link)). The tighter binding of this variant allowed separation of K2_hPg from unbound components during purification. For uniform ¹⁵N- and ¹⁵N/¹³C-K2_hPg, the medium employed was (¹⁵ NH₄)₂SO₄ (99%, Cambridge Isotope Laboratories)/¹³C-glucose (99%; Isotec)/¹³C-methanol (99%, Isotec). The K2_hPg contained seven exogenous residues at the N-terminus of K2_hPg (enumerated as −7 to −1) as a necessary result of the construction of the expression plasmid (Nilsen et al., 1997 ). The final construct is as follows:
$${\text{NH}}_2-{\text{Y}}^{-7}\text{VEFSEE}\left[{\text{C}}^{+1}-\text{K}{2}_{\text{hPg}}-{\text{C}}^{78}\right]{\text{AA}}^{80}$$
The Y⁻⁷VEF is from the polylinker site of the plasmid and SEE represents the linker between K1_hPg and K2_hPg in hPg numbering begins at C⁺ and concludes at C ⁷⁸The integrity of all labeled and unlabeled proteins and peptides was determined by MALDI-TOF mass spectrometry on an Autoflex III spectrometer (Bruker Daltonics). For all uniformly-labeled ¹⁵N- and ¹³C/¹⁵N-peptides, single mass peaks were obtained of the correct molecular weights, indicating complete incorporation of the heavy isotopes.

The structure of DSPE-PEG₂₀₀₀-DTPA was characterized by MS, UV spectroscopy, and by FTIR spectroscopy using a Spectrum One FT-IR, (Pelkin Elmer, Massachusetts, USA) spectrometer. MS was performed on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) Autoflex III spectrometer (Bruker Daltonics, Bremen, Germany). Sample (0.5 µL) were spotted onto an AnchorChip 600/384 (BrukerDaltonics, Bremen, Germany) target microtiter plate (MTP), mixed with a saturated solution of α-cyano-4-hydroxycinnamic acid (0.5 µL), and allowed to crystallize at room temperature. The MS spectra were acquired in the reflector/positive mode with external calibration, using Peptide Calibration Standard II as reference (BrukerDaltonics, Bremen, Germany). MS data analysis was performed by using the FlexAnalysis software (Bruker Daltonics).