Analytica Chimica Acta 703 (2011) 194–203 Contents lists available at ScienceDirect Analytica Chimica Acta jou rn al hom epa ge: www.elsevier.com/locate/aca Implications of partial tryptic digestion in organic–aqueous solvent systems for bottom-up proteome analysis Mark J. Wall, Andrew M.J. Crowell, Gordon A. Simms, Fang Liu, Alan A. Doucette ∗ Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS, Canada B3H 4J3 a r t i c l e i n f o Article history: Received 14 January 2011 Received in revised form 8 July 2011 Accepted 10 July 2011 Available online 5 August 2011 Keywords: Solvent-assisted digestion Trypsin Acetonitrile Rapid digestion Enzyme activity Bottom-up mass spectrometry Shotgun proteomics a b s t r a c t For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent. How- ever, our results demonstrate that tryptic digestion in 80% acetonitrile is less efficient than that of conventional (aqueous) digestion. This is a consequence of decreased enzyme activity beyond ∼40% acetonitrile, increased enzyme autolysis and lower protein solubility in 80% acetonitrile. We observe multiple missed cleavages and reduced concentration of fully cleaved digestion products. Nonetheless we confirm, through room temperature solvent-assisted digestion, a consistent improvement in pro- tein sequence coverage when analyzed by mass spectrometry. These results are explained through the increased number of unique digestion products available for detection. Thus, while solvent-assisted diges- tion has clear merits for proteome analysis, one should be aware of the inefficiency of protein digestion though this protocol, particularly with absolute protein quantitation experiments. © 2011 Elsevier B.V. All rights reserved. 1. Introduction While eliminating the need for all sample processing steps ahead of MS would be ideal, multi-step platforms remain a critical aspect of proteome analysis. Gaining traction to profile complex proteome mixtures, top-down MS provides a direct approach to protein characterization [1]. Nonetheless, bottom-up MS, which adds the necessary step of proteome digestion, is the most widely adopted strategy for proteome analysis. Notwithstanding other critical sample manipulations (fractionation, purification, isotope labelled, etc.), proper proteome digestion can have substantial influence on qualitative as well as quantitative proteome profiling. As a consequence, improvements in the digestion step have been a subject of continued research. As the most widely used protease for bottom-up MS, trypsin is well characterized to cleave after lysine and arginine residues (exceptions noted) [2]. Tryptic peptides are ideal for bottom-up MS, being of suitable size and charge for tandem MS. The conventional protocol for tryptic digestion requires a minimal number of steps; an aqueous protein solution is buffered, disulfides are reduced, and the sample is incubated at 37 ◦ C, typically overnight, with an appro- priate ratio of protein to trypsin (10:1 to 100:1 by mass). While simple, this protocol has considerable room for improvement. ∗ Corresponding author. Tel.: +1 902 494 3714; fax: +1 902 494 1319. E-mail address: Alan.Doucette@dal.ca (A.A. Doucette). With a continued push towards higher throughput, researchers have explored methods to maximize the efficiency of tryptic diges- tion. Summarizing some of the more common approaches, these include gel-assisted digestion [3], immobilized enzymes or sub- strates [4,5], microwave radiation [6], detergents [7], ultrasound [8], elevated pressure [9], non-aqueous solvent systems [10], and combinations thereof [11]. Encompassing the main objectives of these approaches, a loose definition for ‘improved’ digestion is to afford shorter digestion times, to facilitate automation, or to generate a greater number of peptides suitable for MS. Also con- sidering absolute protein quantitation [12], the completeness of digestion is another important aspect. Missed cleavages, though potentially desirable for improved protein sequence coverage [13], will potentially skew results for absolute quantitation, if the degree of digestion is either irreproducible or is unknown [14]. Among the systems to enhance tryptic digestion, solvent- assisted digestion is particularly well studied. First applied to bottom-up MS by Russell et al. in 2001 [10], the strategy bor- rows from a practice in industrial enzymatic processing [15,16]. Here, organic solvents have been shown to enhance or alter the selectivity and/or stability of various enzymes. Extended to proteomics, a motivation to solvent-assisted digestion is the poten- tial for enhanced tryptic activity, or more favourable conditions to digest ‘difficult’ proteins. Through solvent-assisted digestion, numerous studies have demonstrated improved sequence cover- age [17], more rapid digestion (typically <1 h) [10,18] or more complete digestion [18]. Given the mounting evidence, solvent- assisted digestion has clear merit in the field of proteomics. 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.07.025