Improved Coverage of Protein Amino Acid Sequence from Enzymatic Digests with MALDI-TOF MS

Zohra Olumee, Damon C. Barbacci, Gerardo A. Brucker

Stanford Research Systems, Sunnyvale, CA, U.S.A.

Introduction:

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry plays an important role in the detection and characterization of complex mixtures such as the peptide fragments of an enzymatic digest of a protein. However, a common problem is that the peptides detected cover a relatively low percentage of the amino acid sequence of the protein. In this study, we report the use of an electrospray sample preparation for MALDI targets for improved detection of peptides from mixtures and improved amino acid sequence coverage of proteins

Methods and Instrumentation:

In our approach, 10 uM solutions of horse heart myoglobin and cytochrome C are mixed with trypsin (1:20 enzyme to substrate ratio). The digestion process takes place at 37 ⁰C for 10 to 30 minutes and is terminated by the addition of matrix solution (30 mM .alpha-cyano-4-hydroxycinnamic acid). Electrospray sample deposition is used to deliver 20 to 100 nL of the digest solution to the sample plate at a flow rate of 330 nL/min. Sample spots are deposited in as little as 10 seconds. A flat multi-spot sample plate is positioned perpendicular to the needle and acts as the ground electrode. The samples are introduced into a home-built linear time-of-flight mass spectrometer equipped with pulsed extraction.

Preliminary Data:

Previous experiments using electrospray sample preparation for MALDI targets show increased shot-to-shot and sample-to-sample reproducibility compared to the dried-droplet method [1]. Furthermore, homogeneity of the analyte across the sample spot surface is illustrated. We observe a similar effect in our experiments when electrospray is used to prepare MALDI sample spots of tryptic digests of 100 femtomol of myoglobin and cytochrome C. Additionally, the analysis of these digest solutions results in the detection of 21 and 17 peptide fragments from myoglobin and cytochrome C, respectively. The peptides detected cover 100% of the primary amino acid sequence of the respective proteins. In contrast, when a dried droplet sample preparation is prepared from the same digest solutions, only 50% of the amino acid sequence is recovered in the analysis. To our knowledge, this is the first example of such a dramatic improvement in sequence coverage through sample preparation modifications.

Besides the improvement in peptide recovery, there are several more advantages to electrospray sample preparation: (1) electrospray permits consistent sampling of very small volumes (20-100 nL) not typically affordable with the dried droplet method using manual pipettes; therefore, sample volumes may be kept low to increase concentration without sacrificing reproducibility; (2) signal reproducibility from shot-to-shot and homogeneity across the sample surface can enhance the utility of MALDI-TOF for MS/MS experiments; and (3) it is easily automated for high throughput.

Further efforts to characterize the homogeneity of the sample spot, to illustrate the utility for MS/MS experiments, and automation are currently underway.

[1] Hensel, R.R.; King, R.C.; Owens, K.G. Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793.