Simultaneous Quantification of Hundreds of Phosphorylated Metabolites in Multiple Biological Matrices with Ion-Pairing Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry

Many phosphorylated analytes are present in complex mixtures and have diverse essential functions involving biology, chemistry, food, and environmental sciences; their simultaneous quantification is vital for efficiently revealing their crucial functions but remains challenging. Here, we developed a high-coverage method for quantifying many phosphorylated analytes in one run using ion-pairing reversed-phase ultrahigh-performance liquid chromatography and tandem mass spectrometry. Good sensitivity (limit-of-detection <0.95 pmol), linearity (R2 > 0.99), recoveries (80%-120%), precision (CV < 20%), and intertechnician consistency (CV < 20%) were demonstrated for simultaneously quantifying 125 such analytes including nucleotides, nucleotide sugars, phosphorylated amino acids and sugars, and the enzyme-cofactors derived from vitamin B1 (TMP, TPP, TTP), B2 (FMN, FAD), B3 (NAD, NADP), B5 (acyl-CoAs), and B6 (PLP, PMP). The method applicability was also confirmed by quantifying 43-78 phosphorylated metabolites in five typical biological matrices including human urine, plasma, cells, feces, and rabbit liver tissue, illustrating their significant molecular phenotypic differences in the phosphometabolome. By quantifying the phosphometabolomic differences using the method, we further revealed some metabolic characteristics associated with the drug resistances for human non-small cell lung cancer cells. This offers a reliable method for the quantitative investigation of phosphorylated metabolites and their functions.