Automating proteome analysis: improvements in throughput, quality and accuracy of protein identification by peptide mass fingerprinting

Abstract

The use of robots has major effects on maximizing the proteomic workflow required in an increasing number of high-throughput projects and on increasing the quality of the data. In peptide mass finger printing (PMF), automation of steps downstream of two-dimensional gel electrophoresis is essential. To achieve this goal, the workflow must be fluid. We have developed tools using macros written in Microsoft Excel and Word to complete the automation of our platform. Additionally, because sample preparation is crucial for identification of proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we optimized a sandwich method usable by any robot for spotting digests on a MALDI target. This procedure enables further efficient automated washing steps directly on the MALDI target. The success rate of PMF identification was evaluated for the automated sandwich method, and for the dried-droplet method implemented on the robot as recommended by the manufacturer. Of the two methods, the sandwich method achieved the highest identification success rate and sequence coverage of proteins.

Thiophilic adsorption revisited☆

“…Each point on the diagrams corresponds to an independent chromatography. Digester (GE Healthcare) [11]. Protein identification was carried out by peptide mass fingerprinting using a Biflex IV (Bruker Daltonique, Wissembourg, France) [12,13].…”

A proteomic approach to investigate potential biomarkers directed against membrane‐associated breast cancer proteins

“…Digestion was carried out using the Amersham Ettan Digester as previously described [24]. Protein identification was carried out either by PMF using a Biflex IV (Bruker Daltonique, Wissembourg, France), or by nano-LC-MS/MS on Agilent 1100 series IT equipped with a nanospray ion source (Agilent Technologies, Santa Clara, CA, USA) as previously described [19,20].…”

“…Protein identification was carried out either by PMF using a Biflex IV (Bruker Daltonique, Wissembourg, France), or by nano-LC-MS/MS on Agilent 1100 series IT equipped with a nanospray ion source (Agilent Technologies, Santa Clara, CA, USA) as previously described [19,20]. A sandwich spotting method was used for PMF [24] with a-cyano-4-hydroxycinnamic acid (HCCA) as matrix. Using MASCOT Server software package with MASCOT Daemon client application, mass spectra of tryptic digest peptides were searched in batch mode against a downloaded copy of the biweekly updated UniProtKB/SwissProt database (http://www.expasy.ch/sprot/download.html).…”

Automated integration of monolith‐based protein separation with on‐plate digestion for mass spectrometric analysis of esophageal adenocarcinoma human epithelial samples

“…LC/MALDI has also proven useful for unique applications and has been demonstrated to be useful in quantitative studies [14] and in the characterization of post-translational modifications (PTMs) [15] of proteins. In experiments utilizing LC/MALDI and applied to complex biological mixtures, proteins have been exclusively analyzed by enzymatic digestion of whole cell lysates [16][17][18][19][20] using sequential coupling of ion exchange and RP-HPLC separations. Although this shotgun proteomics approach is useful for a comprehensive analysis on a global scale [21], the results can be misleading, where the complexity of samples may result in false positive identifications when only a small number of peptides are matched [22].…”