Combining complementary multiplex extraction chemistries enhances proteome coverage and analytical insights in tiny...

Advancements in protein extraction methodologies are essential for enhancing insect proteomics, especially in sap-feeding small insects, where high lipid content and limited tissue volume impede efficient protein recovery. This study systematically assessed seven extraction chemistries, lysis buffer (LB), dissolution buffer (DB), TRIzol®, trifluoroacetic acid (TFA), trichloroacetic acid (TCA), TCA-phenol, and phenol- under varying pH conditions to determine the most effective workflow for comprehensive proteomic profiling of Bemisia tabaci. Qualitative two-dimensional electrophoresis (2-DE) analysis indicated that phenol and TCA-phenol methods maintained protein integrity across a wide molecular weight range, while acidic conditions enhanced the resolution of low-abundance proteins. Quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses demonstrated that the TCA-phenol method yielded the highest protein recovery and reproducibility (coefficient of variation = 12.6 %), identifying 1010 proteins with balanced representation of soluble and membrane fractions. Principal component and overlap analyses confirmed distinct, method-specific proteomic signatures, with TCA-phenol and TFA achieving the most extensive proteome coverage. Functional annotation revealed that DB and LB enriched primary metabolic and biosynthetic processes, whereas the TCA-phenol method provided the broadest functional spectrum, capturing detoxification, energy metabolism, and stress-related proteins. Overall, this research illustrates that no single extraction chemistry is universally optimal; rather, combining complementary methods enhances proteome coverage. These findings establish a standardized comparative framework for small-insect proteomics and offer valuable methodological insights to advance research in insect physiology, stress adaptation, and ecological interactions.