A peptide concentration and purification method for protein characterization in the subpicomole range using matrix assisted laser desorption/ionization‐postsource decay (MALDI‐PSD) sequencing

Abstract

We here describe the use of added reversed-phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide-bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix-assisted laser desorption/ionization (MALDI)-matrix containing organic/aqueous solvent and transferred to a MALDI-target for mass analysis. We show that by using this bead-peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 microL. We have used this concentration procedure in combination with MALDI-post-source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead-peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI-mass spectrometry (MS) experiments. We have found additionally that the bead-peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead-peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI-MS sample preparation process.

The Effect of α-Amanitin on the Arabidopsis Seed Proteome Highlights the Distinct Roles of Stored and Neosynthesized mRNAs during Germination

“…Spots of interest were excised from GelCode-stained 2D gels and digested by sequence grade trypsin (Promega Biotec, Madison, WI). After digestion, the supernatant containing peptides was concentrated by batch adsorption on POROS 50 R2 beads (Roche Molecular Biochemicals, Basel), and used for MALDI-mass spectrometry analysis on a Bruker Reflex II MALDI-TOF spectrometer after on-target desorption with matrix solution (Gevaert et al, 1998). Before each analysis, the instrument was externally calibrated using two synthetic peptides spotted as near as possible to the biological sample.…”

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation

“…The *30 kDa protein band, present in the tBid-induced supernatant, together with the corresponding region of the lane loaded with control supernatant, were excised from the gel, trypsinized and used for MALDI-MS peptide mass fingerprint analysis. 13 MALDI-PSD analysis of a peptide with a mass of 1370.74 Da, present in the spectrum of the tBid-induced condition but absent in the negative control ( Figure 2B and C), led to the identification of the peptide baring the sequence NH 2 -ASGLLFVPNILAR-COOH from the mouse endonuclease G protein. This was further verified by manually checking the PSD-spectrum for the presence of other tryptic peptide fragments of endonuclease G ( Figure 2D).…”

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid

“…Gel slices were digested using trypsin, as described previously. 73 After digestion, supernatant containing tryptic peptides was removed from the gel pieces and acidified using 1 ml formic acid. A small fraction (10%) of this mixture was concentrated on Poros 50 R2 beads (Roche Molecular Biochemicals, Basel, Switzerland) and used for MALDI-MS peptide mass-fingerprint analysis as previously described.…”

“…Eluting peptides were automatically collected in an aqueous solution containing a small number of Poros 50 R2 beads. 73 These fractions were completely dried in a centrifugal vacuum concentrator and stored at 7208C for further analysis by MALDI-MS.…”