Concentration and desalting of peptide and protein samples with a newly developed C18 membrane in a microspin column format

Abstract

Protein identification plays an important role in today's academic and industrial proteomic research. Commonly used methods for the separation of proteins from complex samples include liquid chromatography (e.g., ion exchange, reversed-phase, hydrophobic interaction), or types of gel electrophoresis (e.g., 1d and 2d PAGE). Relevant proteins separated in the latter way are often cut out, cleaved with trypsin "in gel," and the resulting peptide mixtures combined with matrix and spotted onto a target plate for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-ms) analysis. Subsequently, proteins can be identified by comparison of the resulting peptide mass fingerprints against different databases.(1) since the success of protein identification can be enhanced by the desalting and concentration of the samples, an innovative C18-membrane was incorporated into a microspin column (Vivapure C18 micro spin column, Vivascience AG, Hannover, Germany) to analyze its performance for sample preparation prior to MALDI-ToF-ms. Rapid concentration of single or multiple 200-microl volumes through an available membrane only 2 mm in diameter allowed for analysis of very dilute samples. We observed the successful and rapid desalting of urea-containing protein samples at 100 fmol/mul up to a mass of approximately 70 KDA and the concentration of digest peptides from a solution of 1 fmol/microl using C18-membrane technology.

FIGURE 1

Elutions with 1–4 μl of 50% acetonitrile/0.1% TFA and α-cyano-4-hydroxycinnamic acid versus ion count for angiotensin II (1046) and bombesin (1619) for 1-pmol load.

FIGURE 2

A: MALDI spectrum of 0.5 μl of a <1 fmol/μl BSA digest. B: spectrum of 0.5 μl of the 3-μl eluate after preparation with the Vivapure C18 device. The annotated signals belong to BSA peptides.

FIGURE 3

A. Sypro Ruby–stained SDS-PAGE. +, sample treated with DTT and iodoacetamide, −, sample without treatment. B–D: MALDI spectra: BSA (B); lysozyme without (C) and with (D) alkylation after sample preparation with Vivapure C18 Micro Spin columns.