Jun X. Yan

Protein phosphorylation: technologies for the identification of phosphoamino acids.

Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.

Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate

Abstract An improved high-performance liquid chromatography method for the separation of amino acids derivatised with 9-fluorenylmethyl chloroformate (Fmoc) is described. This method, in conjunction with a multi-tasking program, not only allows high automatic througput but also offers baseline resolution of the common Fmoc-amino acids from a linear acetonitrile gradient, greater chromatographic reproducibility over the life of the column (800 runs) and easy column regeneration. The methods described are shown to be suitable for analysis of hydrolysates of proteins from two-dimensional gels for protein identification purposes, and will also be useful for routine quality control and screening of biological samples.

Large-scale amino-acid analysis for proteome studies

Amino-acid analysis is a relatively new method for identification of proteins separated by two-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. This article describes modified amino-acid analysis methods for this purpose. Streamlined sample handling is a key feature of the process. To minimise sample manipulation, a single vial is used for hydrolysis and the protein hydrolysate on PVDF membrane is extracted by a one-step procedure. The hydrolysate should not be stored for long periods before analysis. Applications of the technique are presented to demonstrate the identification procedure. This approach is the most cost-effective and time-effective first step in mass protein screening for a large-scale proteome project.

Modified immobilized pH gradient gel strip equilibration procedure in SWISS‐2DPAGE protocols

In the present paper we report a revised protocol for immobilized pH gradient (IPG) gel strip equilibration involving a procedural modification between the first‐ and second‐dimensional separation in both analytical and preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). By changing the pH of the equilibration buffer (pH 8.0), the concentration of alkylating reagent (125 mM iodoacetamide) and the time of incubation (15 min), it has been possible to achieve increased cysteine (Cys) alkylation to completion with only one adduct of carboxyamidomethyl‐Cys formed. Importantly, the modification does not alter the 2‐D proteome patterns and therefore maintains the integrity of the existing SWISS‐2DPAGE entries. Results are presented for comparative analyses using human plasma, and for Cys analysis of human albumin to illustrate the advantages of the improved protein reduction and Cys alkylation. The modified step of IPG gel strip equilibration will assist protein digestion for matrix‐assisted laser desorption/ionisation ‐ time‐of‐flight ‐ mass spectrometry analysis, and make Cys quantitation possible without further in‐gel or on‐blot alkylation.

Method for identification and quantitative analysis of protein lysine methylation using matrix-assisted laser desorption/ionization — time-of-flight mass spectrometry and amino acid analysis

Protein methylation is a post‐translational modification that might have important functional roles in cell regulation. We present a new technique with sufficient sensitivity (sub‐pmol level) for analysis of methylation of proteins in abundances typically found on proteome maps produced by two‐dimensional (2‐D) gel electrophoresis. The method involves the identification and quantitation of lysine (Lys) methylation using Fmoc (9‐fluorenylmethyl chloroformate)‐based amino acid analysis (AAA). Tri‐ and monomethyl‐Lys were baseline‐separated from other amino acids using a modified buffer system. Trimethyl‐Lys was quantitatively recovered after acid hydrolysis and AAA of two known methylated proteins — yeast cytochome c and human calmodulin. The methylated peptides from tryptic digestion of those two proteins were identified by high sensitivity matrix‐assisted laser desorption/ionization — time‐of‐flight (MALDI‐TOF) mass spectrometry (MS). An automated mass‐screening approach is proposed for the study of various post‐translational modifications to understand the distribution of those protein isoforms separated by two‐dimensional polyacrylamide gel electrophoresis. It is concluded that the combination of AAA and MALDI‐TOF‐MS provides a high sensitivity quantitative tool for the analysis of protein post‐translational methylation in the context of proteome studies.

Identification and quantitation of cysteine in proteins separated by gel electrophoresis

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 37 degrees C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

Studies of quantitative analysis of protein expression in Saccharomyces cerevisiae

In the present study amino acid analysis is applied to quantitation of Saccharomyces cerevisiae proteome expression. The quantitation levels obtained are compared to data using densitometric analysis of silver or amido black staining and to the theoretical expression level (codon bias) of the identified proteins determined from their amino acid analysis (AAA). The results show that relative volume ratio (%vol) using Melanie II is a better parameter for spot quantitation than relative optical density ratio (%OD), and amino black staining provides good linearity within the range 1—100 pmol protein. However, AAA shows that theoretical expression levels are not well correlated with actual protein expression level, although there is better correlation when isoforms of the expressed protein are identified and included. It is concluded that amino acid analysis provides accurate protein quantitation and has a continuing role in proteome studies in terms of the rapid and inexpensive quantitation of proteins displayed on proteome maps. We do however recognize that in the context of future clinical applications and large‐scale proteome discovery projects, quantitation and post‐translational modification need to be analyzed by “proteomatic” (i.e., proteome automatic bioinformatic analysis directly from the gel) techniques.

High sample throughput phosphoamino acid analysis of proteins separated by one- and two-dimensional gel electrophoresis

Abstract Studies on protein phosphorylation usually involve radiolabelling techniques and visualisation on gels; this limits studies to those on tissues which can be 32 P labelled. With the advent of reproducible micropreparative two dimensional gel electrophoresis, the proteome (protein complement) of a subcellular fraction, cell or tissue can be displayed. We have investigated the sensitivity of limited protein hydrolysis to detect in vivo phosphorylation in proteins blotted from one or two-dimensional polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes. The method uses 9-fluorenylmethyl chloroformate (FMOC) derivatisation chemistry and a modified HPLC AMINOMATE system. Conditions were established for hydrolysis of the PVDF-blotted protein (5.7 M HCl at 110°C for 4 h) which resulted in the recovery of phosphoserine (Ser(P)), phosphothreonine (Thr(P)) and phosphotyrosine (Tyr(P)). The chromatography was carried out on the same system routinely used for amino acid compositional analysis using a gradient elution modified from that used for separation of 16 amino acids. A chromatographic window was designed where all 3 phosphoamino acids are separated with baseline resolution in the order of Ser(P), Thr(P) and Tyr(P), and eluted before the normal protein amino acids. The total separation time is 13 min and includes the elution of excess FMOC, its derivatives and incompletely hydrolysed peptides. The FMOC fluorescence of these three standard phosphoamino acids is linear in the range 10–100 pmol. The technique is shown to be successful in finding phosphoproteins separated by two-dimensional gel electrophoresis. The method is sensitive (30 pmol of a single site phosphorylated protein, 3.8 pmol of Ser(P) detected) and allows automated sample throughput. This presents an option for rapid screening of protein phosphorylation of large numbers of proteins separated by two-dimensional polyacrylamide gel electrophoresis.