Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS

Abstract

In this study, the efficiency of five peptide-extraction methods—acetonitrile (ACN) precipitation, ultrafiltration, C18 solid-phase extraction (SPE), dispersed SPE with mesoporous carbon CMK-3, and mesoporous silica MCM-41—was quantitatively investigated. With 28 tryptic peptides as target analytes, these methods were evaluated on the basis of recovery and reproducibility by using high-performance liquid chromatography-triple-quad tandem mass spectrometry in selected-reaction-monitoring mode. Because of the distinct extraction mechanisms of the methods, their preferences for extracting peptides of different properties were revealed to be quite different, usually depending on the pI values or hydrophobicity of peptides. When target peptides were spiked in bovine serum albumin (BSA) solution, the extraction efficiency of all the methods except ACN precipitation changed significantly. The binding of BSA with target peptides and nonspecific adsorption on adsorbents were believed to be the ways through which BSA affected the extraction behavior. When spiked in plasma, the performance of all five methods deteriorated substantially, with the number of peptides having recoveries exceeding 70% being 15 for ACN precipitation, and none for the other methods. Finally, the methods were evaluated in terms of the number of identified peptides for extraction of endogenous plasma peptides. Only ultrafiltration and CMK-3 dispersed SPE performed differently from the quantitative results with target peptides, and the wider distribution of the properties of endogenous peptides was believed to be the main reason.

High-Recovery Desalting Tip Columns for a Wide Variety of Peptides in Mass Spectrometry-based Proteomics

“…The mesopores of RP hydrophobic materials often irreversible adsorb peptides, decreasing the recovery efficiency in the purification process. 50 Thus, the high recovery in the case of ChocoTip could be a consequence of the enhanced hydrophobicity of the through-pore surface of SPM with embedded St-DVB particles, as well as the suppression of irreversible peptide adsorption at the mesopores of St-DVB particles. To test this hypothesis, we reduced the number of pieces of Empore SDB-XC disk in the SDB-XC-tip from three to one, anticipating a decrease in irreversible peptide adsorption into the mesopores of St-DVB particles.…”

Further investigation of a peptide extraction method with mesoporous silica using high‐performance liquid chromatography coupled with tandem mass spectrometry

“…The digestion of BSA and ovalbumin and the identification and selection of standard tryptic peptides were performed as described in our previous study . The information about these peptides, including sequences, molecular weight (MW), and pI values, can be found in Supporting Information Table S2.…”

Novel nanoporous covalent organic frameworks for the selective extraction of endogenous peptides

“… 8,11 Reversed phase adsorbents-based SPE mainly relies on hydrophobic interaction, which has been increasingly used. 12,13 Based on this, various nanomaterials were developed to the enrichment of endogenous peptides. These nanomaterials include silica nanospheres, 14 carbon nanotubes, 15 silica-carbon composite nanofibers, 16 zeolite nanocrystals, 17 polymeric functionalized nanoparticles, 18 magnetic nanoparticles 19 and so on.…”

“…For eliminating the co-enrichment of proteins, porous materials attracted more and more attentions. Inorganic porous materials, which were represented by mesoporous silica 20 and mesoporous carbon, 12 have demonstrated commendable performance in this field. Recently, metallic organic framework materials (MOFs) 21 and covalent organic framework materials (COFs) 22 were employed in peptidomics analysis and showed great potentials.…”