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.

MALDI-target integrated platform for affinity-captured protein digestion

“…The continuous supply of digestion buffer containing 20% organic solvent ensures the pH to be maintained and that bound trypsin is made available for the digestion process. Since the ISET has the same pitch as a standard 384 microplate, automated sample handling is feasible [37]. The hydrophobicity of the ISET surface achieved by silanization provided a good confinement of 5 – 10 μLfluid volume on each of the ISET nanovial positions.…”

“…The ISET comprises an array of 96 perforated nanovials and is used as a direct interface for MALDI MS. The platform has previously been successfully applied to purify peptides captured by antibodies [36] and automated digestion of HIS-tag purified recombinant proteins [37]. …”

Ambient DESI and LESA-MS Analysis of Proteins Adsorbed to a Biomaterial Surface Using In-Situ Surface Tryptic Digestion

“…Inkjet printing of protein samples and trypsin for surface digestion could offer several advantages over simple surface deposition of the enzyme, as it allows a smaller volume to be used in the analysis, uniformity of spot size, faster turnover via automation, and use within an array that allows high throughput experiments to be performed. MS protein detection from arrays is an emerging field [ 43 , 44 ], and both DESI and LESA-MS have the potential to become useful ambient MS techniques for array based high throughput protein analysis.…”

Advances in coupling microfluidic chips to mass spectrometry

“…Sample pretreatment and preparation can also be integrated on MALDI targets, demonstrated by an integrated selective enrichment target (ISET) platform for high‐throughput sample pretreatment and preparation (Ekström et al, 2004, 2006, ; Yan et al, ; Adler et al, ). ISET is a MALDI target with integrated nanocolumns in a 48‐ or 96‐array format, which is compatible with standard liquid handlers and allows the user to load different types of chromatographic media.…”

“…Compared to ZipTip and MassPREP PRO target sample preparation, the ISET‐based SPE sample preparation showed a superior performance with respect to number of detected peptides and their signal intensity. Other application includes packing the nanocolumns with 500 nL of HisPur cobalt resin (Adler et al, ). Purification by immobilized metal ion affinity chromatography (IMAC) and on‐bead tryptic digestion were performed directly in the ISET nanocolumns, enabling peptide mass fingerprinting and MS/MS analysis of the generated peptides.…”