On-line protein digestion by immobilized enzyme microreactor capillary electrophoresis-mass spectrometry

• Published in: Talanta (Oxford) 2019-07, Vol.199, p.116-123
• Main Authors: Villegas, Lorena, Pero-Gascon, Roger, Benavente, Fernando, Barbosa, José, Sanz-Nebot, Victoria
• Format: Article
• Language: English
• Subjects: Bottom-up proteomics; Capillary electrophoresis; Caseins - chemistry; Caseins - metabolism; Electroforesi capil·lar; Electrophoresis, Capillary; Enzims; Enzymes; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Espectrometria de masses; Humans; Immobilized enzyme; Lactoglobulins - chemistry; Lactoglobulins - metabolism; Mass Spectrometry; Microreactor; On-line digestion; Trypsin - chemistry; Trypsin - metabolism
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2019.02.039

In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting β-lactoglogulin (β-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, on-line digestion, separation and characterization of the protein digests were possible in less than 30 min. The limit of detection for complete sequence coverage was around 10 µg mL−1 (~500 µM) of β-LG, the repeatability was comparable to the off-line digestion methods and the microreactor could be reused until thirty times. The good performance of IMER-CE-MS was also demonstrated for several other proteins as α-casein (α-CSN), β-casein (β-CSN), and κ-casein (κ-CSN), as well as for a complex protein mixture (an Escherichia coli whole cell lysate). [Display omitted] •Microreactors are packed with immobilized trypsin particles for on-line IMER-CE-MS.•On-line bottom-up analysis of proteins is completed in less than 30 min.•Performance is comparable to off-line digestion with free or immobilized trypsin.•The potential is demonstrated for standard proteins and an E. Coli whole cell lysate.