Advanced

Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS.

Adler, Belinda LU ; Boström, Tove; Ekström, Simon LU ; Hober, Sophia and Laurell, Thomas LU (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:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Chemistry
volume
84
issue
20
pages
8663 - 8669
publisher
The American Chemical Society
external identifiers
  • pmid:22971087
  • wos:000309805200034
  • scopus:84869486988
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
2012-10-19 10:40:00
date last changed
2017-01-01 03:49:54
@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},
  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},
  volume       = {84},
  year         = {2012},
}