Reducing Cathodic Drift during Isoelectric Focusing Using Microscale Immobilized pH Gradient Gels.
Anal Chem
; 96(21): 8648-8656, 2024 05 28.
Article
en En
| MEDLINE
| ID: mdl-38716690
ABSTRACT
Microfluidic analytical tools play an important role in miniaturizing targeted proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (µIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5 mm separation lane over a 20 min focusing duration. During the 20 min duration, we observed markedly different cathodic drift velocities among the three formulations 60.1 µm/min in CA-IEF, 2.5 µm/min in IPG-IEF (â¼24-fold reduction versus CA-IEF), and 1.4 µm/min in mixed-bed IEF (â¼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. µIPG is a promising and stable technique for studying proteoforms from small volumes.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Dimetilpolisiloxanos
/
Focalización Isoeléctrica
Límite:
Humans
Idioma:
En
Revista:
Anal Chem
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos