Dataset Information

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS.

ABSTRACT: Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm×150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC-MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 ?m×150 mm column was used online and the online elution gradients for each fraction were adjusted in order to obtain well resolved separation. Comparing this method directly to only performing nanoLC-MS we observed a 61.6% increase in the number of identified proteins. At a 1% false discovery rate 1028 proteins were identified using two dimensions of RPLC versus 636 proteins identified in a single nano-flow separation. The majority of proteins identified by one dimension of nano-LC were present in the proteins identified in our two dimensional strategy. Although increasing analysis time, this non-orthogonal and facile pre-fractionation method affords a more comprehensive examination of the proteome.

SUBMITTER: Gokce E

PROVIDER: S-EPMC3703961 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS.

Gokce Emine E Andrews Genna L GL Dean Ralph A RA Muddiman David C DC

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 20110204 9-10

Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm×150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC-MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 μm×150 mm column was used online and the o ...[more]

PMID: 21342794

Similar Datasets

Project description:BACKGROUND:Wheat grain avenin-like proteins (ALPs) belong to a recently discovered class of wheat grain storage protein. ALPs in wheat grains not only have beneficial effects on dough quality but also display antifungal activities, which is a novel observation for wheat storage proteins. Previous studies have shown that ALPs are likely present in the albumin/globulin fractions of total protein extract from wheat flour. However, the accumulation characteristics of these ALPs in the mature wheat grain remains unknown. RESULTS:In the present study, a total of 13 ALPs homologs were isolated and characterized in the albumin/globulin fractions of the wheat protein extract. A combination of multiple techniques including RP-HPLC, SDS-PAGE, MALDI-TOF and peptide sequencing were used for accurate separation and identification of individual ALP homolog. The C-terminal TaALP-by-4AL/7DS, TaALP-by-4AL/7AS/7DS, TaALP-bx/4AL/7AS/7DS, TaALP-ay-7DS, TaALP-ay-4AL, TaALP-ax-4AL, TaALP-ax-7AS, and TaALP-ax-7DS, were separated as individual protein bands from wheat flour for the first time. These unique ALPs peptides were mapped to the latest wheat genome assembly in the IWGSC database. The characteristic defence related proteins present in albumin and globulin fractions, such as protein disulfide-isomerase (PDI), grain softness protein (GSP), alpha-amylase inhibitors (AAIs) and endogenous alpha-amylase/subtilisin inhibitor were also found to co-segregate with these identified ALPs, avenin-3 and α-gliadins. The molecular weight range and the electrophoresis segregation properties of ALPs were characterised in comparison with the proteins containing the tryp_alpha_amyl domain (PF00234) and the gliadin domain (PF13016), which play a role in plant immunity and grain quality. We examined the phylogenetic relationships of the AAIs, GSP, avenin-3, α-gliadins and ALPs, based on the alignment of their functional domains. MALDI-TOF profiling indicated the occurrence of certain post-translations modifications (PTMs) in some ALP subunits. CONCLUSIONS:We reported for the first time the complete profiling of ALPs present in the albumin/globulin fractions of wheat grain protein extracts. We concluded that majority of the ALPs homologs are expressed in wheat grains. We found clear evidence of PTMs in several ALPs peptides. The identification of both gliadin domain (PF13016) and Tryp_alpha_amyl domain (PF00234) in the mature forms of ALPs highlighted the multiple functional properties of ALPs in grain quality and disease resistance.

Dataset Information

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS.

Publications

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets