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A versatile sample pretreatment method for the honey matrix is still needed for any proteomic-based investigations. Invertase and diastase are the most important enzymes in the maturation of pine honey and the origin of these enzymes are attributed to the bee\"s hypopharyngeal glands (HPG). In our study, we aimed to isolate and enrich these enzymes as model proteins representing the honey proteome in an efficient and practical way. As authenticity comparison, isolating the same enzymes from HPG samples was also accomplished. For yielding pine honey crude protein isolate, as a tandem two-step approach, stirred cell ultrafiltration followed by centrifugal ultrafiltration (CUF) protocol was determined after experimental optimization. HPGs were dissected from the Apis mellifera L. and proteins were extracted by using a bead beater followed by concentration using CUF. Protein profiles of pine honey and HPG were compared by SDS-PAGE. The resulting protein concentrations, enzyme activities, and the cleaning efficiencies of the applied techniques were evaluated and optimized using the Bradford assay, modified enzyme activity assays, and sugar profiling method developed at HPLC-RID. The novel pretreatment method provided invertase at 1055.1 U/kg activity and diastase at 693.3 Shade U/g activity with yields of 900.9% and 2432.6%, respectively. The final crude protein isolate can be interpreted as reference material at any authenticity and quality assays of honey. The obtained crude protein extract will pave the way for high throughput proteomic investigations at the honey matrix. Furthermore, this template methodology could be scaled up in the industry for natural enzyme production.

Streptomycin (STR) and dihydrostreptomycin (DSTR) are the typically encountered aminoglycoside (AMG) residues in honey. For AMG analysis, studies in literature involve impractical and expensive applications such as ion-pairing chromatography, immunoassays, pre and post column derivatizations, or SPE approaches. Pre -treatments of these methods are toilsome and costly. Herein, one-pot, aqueous in-situ derivatization method was presented as a superior protocol. Time and cost-efficient UHPLC-MS/MS method has been developed, and practical sample preparation was introduced. Satisfactory results were reported in method verification studies. The mean recovery values were 102.6% for STR and 101.3% for DSTR. Average values between 1.5% and 9.9% RSDs were found at intra and inter-day precisions. CC alpha (5.7 and 5.8 mu g/kg) and CC beta (6.2 and 6.4 mu g/kg) values were calculated for STR and DSTR respectively. AMG residues were found in 29 out of 110 analyzed samples using validated method. Described novelty enabled comprehensive analysis in an inexpensive and straightfor-ward manner.