Search

Direct molecular analysis of tissue samples is a promising approach to increase the accuracy, speed and molecular specificity of cancer diagnosis. Herein, alterations of amino acids between human lung cancer tissues and matched adjacent normal tissues were profiled by internal extraction electrospray ionization mass spectrometry (iEESI-MS). The results indicated that the levels of 11 detected amino acids (including serine, proline, valine, threonine, asparagine, aspartic acid, methionine, histidine, phenylalanine, arginine and tyrosine) in the cancerous tissues were lower than that in the adjacent normal tissues. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA) model, cancerous and adjacent normal tissues were clearly discriminated, and the amino acids that played the major role in the differentiation between cancerous and adjacent normal tissues were identified. Moreover, metabolic pathway analysis revealed alterations of differential amino acids in several metabolic pathways upon lung cancer. The current study extends the power of iEESI-MS as a promising tool for quantitative characterization of amino acids in tissues, and allows the study of alterations in amino acids metabolism associated with the development of lung cancer.

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.