Proteomic Profiling and Protein Identification by MALDI-TOF Mass Spectrometry in Unsequenced Parasitic Nematodes

Paul Millares,Mark C Prescott,Samirah Perally,Peter M Brophy,Huw H Rees,Deborah A Ward,E James Lacourse,Jane E Hodgkinson

doi:10.1371/journal.pone.0033590

Abstract

Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This study demonstrates the cost-effective use of available EST databases and inexpensive, accessible MALDI-TOF MS in conjunction with PMF for reliable protein identification in unsequenced organisms.

Highlights

The identification of proteins in proteomic studies requires either (i) Tandem Mass Spectrometry (MS/MS) [1], which is relatively expensive, followed by either ion searches of databases or limited de novo sequencing of peptides [2] before database searching; or (ii) Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS) [3], followed by Peptide Mass Fingerprint (PMF) searching of databases [4]
Analyses by Progenesis PG200 v2006 software (Non-Linear Dynamics) allowed all the spots derived from the differently loaded gels to be matched. 602 spots were common to all differently loaded gels. 100 abundant spots were selected according to their normalised volumes, and were processed for further identification by PMF using MALDI-TOF MS (Figure 1)
The MALDI-TOF mass spectra resulting from the 100 tryptically digested spots were processed using ProteinLynx software

Summary

Introduction

The identification of proteins in proteomic studies requires either (i) Tandem Mass Spectrometry (MS/MS) [1], which is relatively expensive, followed by either ion searches of databases or limited de novo sequencing of peptides [2] before database searching; or (ii) Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS) [3] (significantly less expensive than MS/MS), followed by Peptide Mass Fingerprint (PMF) searching of databases [4]. Gastro-intestinal nematode infections involving parasites such as Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp. or Nematodirus spp. pose a great health and economic burden to the sheep industry worldwide [5,6,7]. H. contortus is a highly pathogenic parasite of small ruminants, posing a significant risk to animal health and productivity worldwide and as such is a major focus of anthelmintic resistance studies [11,12]

Methods

Results

Conclusion