Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS.
(2012) In Analytical Chemistry 84(20). p.8663-8669- Abstract
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/3124005
- author
- Adler, Belinda LU ; Boström, Tove ; Ekström, Simon LU ; Hober, Sophia and Laurell, Thomas LU
- organization
- publishing date
- 2012
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Analytical Chemistry
- volume
- 84
- issue
- 20
- pages
- 8663 - 8669
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:22971087
- wos:000309805200034
- scopus:84869486988
- pmid:22971087
- ISSN
- 1520-6882
- DOI
- 10.1021/ac3017983
- language
- English
- LU publication?
- yes
- id
- e2ad1516-6556-4a0d-a3d2-7175e7154fa1 (old id 3124005)
- date added to LUP
- 2016-04-01 10:42:20
- date last changed
- 2022-01-26 01:41:32
@article{e2ad1516-6556-4a0d-a3d2-7175e7154fa1, abstract = {{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.}}, author = {{Adler, Belinda and Boström, Tove and Ekström, Simon and Hober, Sophia and Laurell, Thomas}}, issn = {{1520-6882}}, language = {{eng}}, number = {{20}}, pages = {{8663--8669}}, publisher = {{The American Chemical Society (ACS)}}, series = {{Analytical Chemistry}}, title = {{Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS.}}, url = {{http://dx.doi.org/10.1021/ac3017983}}, doi = {{10.1021/ac3017983}}, volume = {{84}}, year = {{2012}}, }