title: Miniaturized and automated high-throughput verification of proteins in the ISET platform with MALDI MS.
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.
authors.affiliation: Department of Measurement Technology and Industrial Electrical Engineering, Division of Nanobiotechnology, Lund University, Box 118, SE-211 00 Lund, Sweden.
authors.forename: Belinda
authors.initials: B
authors.lastname: Adler
authors.forename: Tove
authors.initials: T
authors.lastname: Boström
authors.forename: Simon
authors.initials: S
authors.lastname: Ekström
authors.forename: Sophia
authors.initials: S
authors.lastname: Hober
authors.forename: Thomas
authors.initials: T
authors.lastname: Laurell
mesh_terms: D000595:Amino Acid Sequence; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods*; D006801:Humans; D008904:Miniaturization; D010449:Peptide Mapping / Q000191:economics / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods*; D013997:Time Factors
chemical_list: D011506:Proteins; D011994:Recombinant Proteins