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The type I cGMP-dependent protein kinases serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMPdependent protein kinase (PKA). Here, we examined PKG Ia activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 ( 53). We found that the construct exists as a monomer as assessed by whole-protein mass spectrometry, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that 53 has a similar shape to the heterodimeric RIa:C complex of PKA. Moreover, we found that the 53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length as assessed by surface plasmon resonance spectroscopy (SPR). However we found that the 53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Ia activation is distinct from PKA, and its cooperativity is driven by in trans interactions between protomers.

Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm2), followed by parallel accumulation–serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min gradient. This result corresponded to ∼19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single-range TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by the average and median coefficient of variation values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.

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