Automated integration of monolith‐based protein separation with on‐plate digestion for mass spectrometric analysis of esophageal adenocarcinoma human epithelial samples

Abstract

A unique approach of automating the integration of monolithic capillary HPLC-based protein separation and on-plate digestion for subsequent MALDI-MS analysis has been developed. All liquid-handling procedures were performed using a robotic module. This automated high-throughput method minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and analysis. Also, precise positioning of the droplet from the capillary HPLC separation onto the MALDI plate allows for preconcentration effects of analytes for improved sensitivity. Proteins from primary esophageal Barrett's adenocarcinoma tissue were prefractionated by chromatofocusing and analyzed successfully by this automated configuration, obtaining rapid protein identifications through PMF and sequencing analyses with high sequence coverage. Additionally, intact protein molecular weight values were obtained as a means to further confirm protein identification and also to identify potential sequence modifications of proteins. This simple and rapid method is a highly versatile and robust approach for the analysis of complex proteomes.

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

“…Recently, a number of methods have been developed with integrate protein separation by monolithic LC with on‐plate tryptic digestion for subsequent analyses by MALDI‐time‐of‐flight (TOF)‐MS and MALDI‐TOF/TOF‐MS (Yoo et al, 2006; Zheng et al, 2006). This helped to reduce the amount of time required for traditional procedures involving in‐solution enzymatic digestion and sample cleanup prior to MALDI‐MS analysis.…”

Separation of intact proteins by using polyhedral oligomeric silsesquioxane based hybrid monolithic capillary columns

“…Therefore, the separation selectivity of the POSSbased hybrid monolithic column can be adjusted by combining different types of function monomers to some extent, which may be an alternative method to improve the LC separation capability. The PS-DVB monolithic column has been widely used in the purification, fractionation and LC separation of the intact proteins [30][31][32][33][34]. Thus, a commercial PS-DVB monolithic capillary column has also been investigated for comparison (Fig.…”

Enhanced protein identification using graphite-modified MALDI plates for offline LC-MALDI-MS/MS bottom-up proteomics

“…The sample can be modified after separation with techniques such as on-plate digestion. 13,14,15 Also, data-dependent analysis can be performed without duty-cycle restrictions allowing for the manipulation of precursor ion selection strategies to improve the efficiency of MS/MS acquisition. 5,16,17,18 Fractions can also be analyzed in an alternating fashion to gain complementary 19 or additional 20,21 information.…”