Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
2012 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 20, 8663-8669 p.Article in journal (Refereed) Published
A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.
Place, publisher, year, edition, pages
2012. Vol. 84, no 20, 8663-8669 p.
Assisted-Laser-Desorption/Ionization, Selective Enrichment Target, Spectrometry-Based Proteomics, Mass-Spectrometry, Sample Preparation, Affinity-Chromatography, Recombinant Proteins, Microfluidic Chips, Identification, Expression
IdentifiersURN: urn:nbn:se:kth:diva-107629DOI: 10.1021/ac3017983ISI: 000309805200034ScopusID: 2-s2.0-84869486988OAI: oai:DiVA.org:kth-107629DiVA: diva2:577775
FunderKnut and Alice Wallenberg FoundationSwedish Research Council, VR 2009-5361 VR/Vinnova/SSF MTBH 2006-7600Vinnova, 2007-02614
QC 201212172012-12-172012-12-142014-10-22Bibliographically approved