Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.     

Linear multidimensional liquid chromatography in the preparative scale purification of calmodulin from brain extract

Rapid preparative scale purification of calmodulin from crude bovine brain extract is achieved in a single chromatographic run by physically coupling two different liquid chromatography columns which employ different separation mechanisms. In this case columns packed with newly commercialized 40-microns silica-based hydrophobic interaction and 5-microns micron silica-based weak anion-exchange chromatography media were used. The only sample preparation required for conducting this purification procedure is the addition of salt to the crude brain supernatant to promote the initial binding of calmodulin to the hydrophobic interaction chromatography media. Chromatography carried out on such linear arrangements of columns has been referred to as linear multidimensional liquid chromatography.     

Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC)

A systematic approach to the design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon-alpha2a is used as a model system to determine the operational parameters in membrane-based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen-antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, have been purified efficiently employing membrane-based immunoaffinity chromatography. Overall, membrane-based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial-scale production of recombinant proteins. (c) 1992 John Wiley & Sons, Inc.     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Separation and purification of individual neurofilament proteins by reverse-phase high-performance liquid chromatography

A reverse-phase HPLC method was developed to separate individual neurofilament proteins (210,000, 160,000, and 70,000 Da) from the glial fibrillary acid protein. It is useful for analytical or preparative methods, with yields higher than 80%. The method represents improvement over previous methods in speed, efficiency, and purity. Combining this HPLC method with the conventional chromatographic method on DEAE-cellulose, highly purified individual neurofilament proteins can be obtained in large scale.     

Rapid purification of bacterially expressed fusion proteins by high-performance liquid chromatography methods

Recent developments in recombinant DNA technology allow the high-level expression in bacteria of substantial amounts of viral and eukaryotic proteins whose genes have been cloned into plasmids. The present study reports two high-performance liquid chromatography (HPLC) methods for the rapid purification to apparent homogeneity of these bacterially expressed proteins. The two methods are anion exchange HPLC in the presence of 7 M Urea and reverse-phase HPLC of protein solubilized by 7.0 M guanidine hydrochloride. The two methods have been used successfully to purify fusion products of the v-myb oncogene and fusion proteins from HTLV-I Px and transmembrane regions and should be of general utility for purification of other bacterially produced proteins.     

Rapid screening and scale-up of ultracentrifugation-free,membrane-based procedures for purification of His-tagged membrane proteins

This study develops procedures to rapidly screen conditions for purification of membrane proteins (MPs) using 96-well plates containing nickel-functionalized membranes. In addition to their application in the pharmaceutical industry, MPs are important components of new sensors, synthetic membranes, and bioelectronic devices. However, purification of MPs is challenging due to their hydrophobic exterior, which requires stabilization in amphipathic detergent micelles. We examined the extent of extraction of the light-driven sodium transporter, Krokinobacter eikastus rhodopsin 2 (KR2) heterologously expressed in Escherichia coli using different salts and maltoside-based detergents. The extraction was followed by subsequent affinity purification in membranes functionalized with Ni²⁺-nitrilotriacetate complexes that bind the His-tagged KR2. We also employed a hydrophobic chelator to separate detergent micelles from the aqueous phase as an initial isolation step prior to affinity purification. Unlike conventional resin-based capture, which can take a full day or more, the membrane-based screening of purification conditions takes only a few hours, and its scale-up involves changing from a 96-well format to a larger membrane module. The novelty of the method lies in utilizing membrane-based ultracentrifugation-free purification of MPs from cell membrane fragments; the optimized purification conditions from the screening method can potentially be applied to large-scale/conventional resin-based purification of MPs.     

Towards multidimensional liquid chromatography separation of proteins using fluorescence and isotope-coded protein labelling for quantitative proteomics

HPLC has emerged as a valuable tool for separating proteins. To address the analysis of complex proteomes and quantitative changes of proteins therein, we developed a multidimensional LC (MDLC)-based approach followed by large gel 1-D SDS-PAGE. Here we present a novel strategy that allows for simultaneously identifying and quantifying differentially regulated proteins following three separation and fractionation steps. This MDLC platform integrates advantages of dual protein labelling using both fluorescence and isotope-coded tags for subsequent detection and quantification of abundance ratios of proteins by MS.     

Large scale depletion of the high-abundance proteins and analysis of middle- and low-abundance proteins in human liver proteome by multidimensional liquid chromatography

An unbiased method for large-scale depletion of high-abundance proteins and identification of middle- or low-abundance proteins by multidimensional LC (MDLC) was demonstrated in this paper. At the protein level, the MDLC system, coupling the first dimensional strong cation exchange (SCX) chromatography with the second dimensional RP-HPLC, instead of immunoaffinity technology, was used to deplete high-abundance proteins. Sixty-two fractions from SCX were separated further by RPLC. UV absorption spectra were observed to differentiate high-abundance proteins from middle- or low-abundance proteins. After the depletion of high-abundance proteins, middle- or low-abundance proteins were enriched, digested, and separated by online 2D-micro-SCX/cRPLC. The eluted peptides were deposited on the MALDI target and detected by MALDI-TOF/TOF MS. This depletion strategy was applied to the proteome of the normal human liver (NHL) provided by the China Human Liver Proteome Project (CHLPP). In total, 58 high-abundance proteins were depleted in one experiment. The strategy increases greatly the number of identified proteins and around 1213 proteins were identified, which was about 2.7 times as that of the nondepletion method.     

Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins).

The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein.     

Development of multidimensional liquid chromatography and application in proteomic analysis

As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.     

Development of multidimensional liquid chromatography and application in proteomic analysis

As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.     

Novel strategy of high-abundance protein depletion using multidimensional liquid chromatography

In this study, for the first time, a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of specificity depletion method, was developed at the intact protein level for depletion of high-abundance proteins from rat liver. Proteins were prefractionated by SCX in the first dimensional separation, followed by RPLC with high resolution separation. UV absorption intensity was used to differentiate high-abundance proteins. The proteins with the absorbance intensity above 0.1 AU were defined as high abundance proteins and depleted. After removal of high-abundance proteins; other proteins were pooled, digested, and subsequently separated by capillary liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry analysis. The high efficiency of the strategy was demonstrated by analyzing the soluble protein extracted from rat liver tissue. In total, 77 high-abundance proteins were depleted in one experiment flow. The ratio of depleted content of high-abundance proteins to that of total proteins was about 34.5%. In total, 1530 proteins were identified using the depletion strategy. Quantitative estimation of high-abundance proteins through liquid chromatography combined with UV absorption spectra was achieved. On the basis of the reproducible experimental results, a rapid and high-throughput depletion protocol was put forward. Along with depletion of the most (79.1%) high-abundance proteins and the separation of digested peptides, the total separation time could be less than 30 h. This strategy has no bias for depleting high-abundance proteins and enhances the number of identified proteins; therefore, it can be widely used in the global proteins analysis.     

High-performance liquid chromatography of proteins by gel permeation chromatography

The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.     

Profiling of myelin proteins by 2D-gel electrophoresis and multidimensional liquid chromatography coupled to MALDI TOF-TOF mass spectrometry

The myelin sheath is an electrically insulating layer that consists of lipids and proteins. It plays a key role in the functioning of the nervous system by allowing fast saltatory conduction of nerve pulses. Profiling of the proteins present in myelin is an indispensable prerequisite to better understand the molecular aspects of this dynamic, functionally active membrane. Two types of protein, the myelin basic protein and the proteolipid protein, account for nearly 85% of the protein content in myelin. Identification and characterization of the other "minor" proteins is, in this respect, a real challenge. In the present work, two proteomic strategies were applied in order to study the protein composition of myelin from the murine central nervous system. First, the protein mixture was separated by 2D-gel electrophoresis and, after spot excision and in-gel digestion, samples were analyzed by mass spectrometry. Via this approach, we identified 57 protein spots, corresponding to 38 unique proteins. Alternatively, the myelin sample was digested by trypsin and the resulting peptide mixture was further analyzed by off-line 2D-liquid chromatography. After the second-dimension separation (nanoLC), the peptides were spotted "on-line" onto a MALDI target and analyzed by MALDI TOF-TOF mass spectrometry. We identified 812 peptides by MALDI MS/MS, representing 93 proteins. Membrane proteins, low abundant proteins, and highly basic proteins were all represented in this shotgun proteomic approach. By combining the results of both approaches, we can present a comprehensive proteomic map of myelin, comprising a total of 103 protein identifications, which is of utmost importance for the molecular understanding of white matter and its disorders.     

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.     

High-performance liquid chromatography purification of chemically modified RNA aptamers

2′-Fluoro modified RNAs are useful as potential therapeutics and as special substrates for studying RNA function. 2′-Fluoro modified RNAs generally need to be purified after they are prepared either enzymatically or by solid-phase synthesis. Here we introduce a protocol by which 2′-fluoro modified RNAs with 57 and 58 nucleotides can be resolved and purified using ion-pair, reverse-phase high-performance liquid chromatography (HPLC). Because the size of our RNA samples is in the range of many known RNA aptamers of therapeutic values, our protocol should be generally useful.