Journal of Chromatography A, 1111 (2006) 175–191 Exploring human plasma proteome strategies: High efficiency in-solution digestion protocol for multi-dimensional protein identification technology Qinhua Cindy Ru a,∗ , Luwang Andy Zhu a , Richard A. Katenhusen a , Jordan Silberman a , Henry Brzeski a , Michael Liebman a , Craig D. Shriver b a Windber Research Institute, 600 Somerset Ave., Windber, PA 15963, USA b Walter Reed Army Medical Center, 6900 Georgia Ave., NW, Washington, DC 20307, USA Available online 26 July 2005 Abstract Multi-dimensional protein identification technology (MudPIT) is becoming a prevalent proteomic approach due to its high-throughput separations and accurate mass detection. Prior to MudPIT analysis, complicated samples required in-solution digestion. Unlike in-gel digestion, in which enzymes work on just a few proteins, in-solution digestion involves simultaneous digestion of hundreds or thousands of proteins. In-solution digestion protocols must therefore be very efficient. Few investigations have evaluated the efficiency of in-solution digestion protocols. The present research compared three such protocols. Results suggest that a protocol utilizing trifluoroethanol (TFE) as denaturant is most efficient. © 2005 Elsevier B.V. All rights reserved. Keywords: Human plasma proteome; In-solution digestion; Multi-dimensional protein identification technology; Human plasma; Clinical proteomics 1. Introduction Multi-dimensional protein identification technology (MudPIT) is becoming a prevalent proteomic approach [1–3] because it offers high-throughput two-dimensional liquid chromatography (2D LC) separation, accurate tandem mass spectrometry (MS/MS) detection, and reproducible protein identification. Complicated samples must be digested prior to MudPIT analysis. Unlike in-gel digestion, in which en- zymes work on just a few proteins, in-solution digestion in- volves simultaneous digestion of hundreds or thousands of proteins. In-solution digestion protocols must therefore, be very efficient. Interruption of salts from in-solution diges- tion causes an additional problem. In-gel digestion is always followed by desalting and concentration, whereas in-solution digestion is followed by MudPIT analysis. Although the first- dimensional ion exchange separation can desalt the sample, some salts from the digestion may still enter the mass spec- ∗ Corresponding author. Tel.: +1 814 467 9844; fax: +1 814 467 6334. E-mail address: c.ru@wriwindber.org (Q.C. Ru). trometer and interrupt peptide detection. A high-efficiency and low-salt background is therefore essential for carrying out useful MudPIT analyses. Little research [4–7] has compared digestion protocols, especially for in-solution digestion. Few strategies can quan- titatively determine the efficiency of trypsin digestion on a proteome-wide scale [4]. Veenstra et al. [4] developed a method to evaluate the efficiency of in-solution digestion by counting missed cleavage sites in peptides of isotope-labeled yeast. This approach is, of course, not applicable to clinical samples such as human serum, plasma, urine, or tissue. Keller et al. [5] investigated digestion protocols for nanoliter sam- ples, while Hancock et al. [6] and Sutton et al. [7] completed investigations of multiple protease digestion protocols. An effective and reproducible in-solution digestion proto- col will facilitate high- throughput analysis. We have tested several in-solution digestion protocols using the same human plasma sample. Results suggested that, (1) different protocols yield varied results, (2) traditional protocols using urea as denaturant may significantly impede the mass spectrometric detection, and (3) digests from protocols with non-salt type 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.06.080