Protein identification by syringe pump-driven reversed-phase LC-MS/MS

In typical mass spectrometry-based protein identification using peptide fragmentation fingerprinting, front-end separation plays a critical role in successful peptide sequencing. This separation step demands a great deal of time and usually is the rate-limiting step for the whole process. Here we provide an alternative separation method, based on a simple nanoflow delivery system, that is able to shorten the separation time considerably. This system consists of a 25-mul syringe connected to a manually packed reversed-phase mini-capillary column that can be directly coupled to an electrospray ionization tandem mass spectrometer. A syringe pump is then used to deliver the peptide mixtures at a nanoscale flow rate. We examined the efficiency and efficacy of this method by analyzing the tryptic peptides of bovine serum albumin and of 10 Escherichia coli proteins separated by two-dimensional gel electrophoresis (2DE). The results showed that identification of each protein could be achieved successfully within 25 min by using the disposable mini-capillary column. Moreover, all 2DE-separated E. coli proteins were identified at high confidence levels. Together, our data suggest that this method is a suitable option for mass spectrometry-based protein identification.     

A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification

A two-dimensional liquid-phase separation scheme coupled with mass spectrometry (MS) is presented for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid-phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) as the second dimension. Protein quantitation and mass measurement are performed using electrospray ionization-time of flight MS (ESI-TOF MS). Proteins are identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS) and MALDI-quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS). Two pH regions with 50-60 unique proteins in each pH range were chosen for analysis. Mass maps were created that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development.     

Gel‐free mass spectrometry analysis of Drosophila melanogaster heads

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under‐represented group in proteomics datasets. Herein, a bottom‐up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem‐mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter‐aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed‐phase (RP) LC at high pH in the first dimension and acidic RP‐LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 ( http://proteomecentral.proteomexchange.org/dataset/PXD001712 ).     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

High-throughput characterization of lipopolysaccharide-binding proteins using mass spectrometry

Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the "untargeted" mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.     

Comprehensive two-dimensional liquid chromatography mass spectrometric profiling of the rat hippocampal proteome

In this study, we performed the first high‐throughput and comprehensive proteomic profiling of the rat hippocampal proteome. Using a combination of 2‐D LC‐MS and data analysis with the Rosetta Elucidator^® system, we identified 1340 unique proteins. Functional classification showed that most of these were associated with synaptic function and comprised a high proportion of phosphorylated proteins and analytically challenging classes of membrane proteins such as ion channel receptor subunits.     

Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry

In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.     

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.     

Characterization and quantification of karlotoxins by liquid chromatography–mass spectrometry

Karlodinium veneficum is a cosmopolitan dinoflagellate with a worldwide distribution in mesohaline temperate waters. The toxins from K. veneficum, or karlotoxins (KmTxs), which have been implicated in fish kill events, have been purified from monoalgal cultures, and shown to possess hemolytic, cytotoxic and ichthyotoxic activities. Three karlotoxins (KmTx 1–1, KmTx 1–3 and KmTx 2) have been isolated from two different North American strains of K. veneficum and characterized using liquid chromatography–mass spectrometry (LC–MS). KmTx 1 karlotoxins have a UV absorption maximum (λ_max 225 nm) at lower wavelengths than KmTx 2 karlotoxins (λ_max 235 nm). The exact masses and predicted empirical formulae for the karlotoxins (KmTx 1–1, 1308.8210, C₆₇H₁₂₀O₂₄; KmTx 1–3, 1322.8637, and C₆₉H₁₂₆O₂₃; KmTx 2, 1344.7938, C₆₇H₁₂₁ClO₂₄) were determined using high resolution mass spectrometry. Although the individual toxins produce a single peak in reverse phase high performance liquid chromatography (HPLC), MS revealed congeners co-eluting within each peak. These congeners could be separated under normal phase chromatography and revealed a single hydroxylation being responsible for the mass differences. Multistage MS (MSⁿ) showed that the three KmTxs and their congeners share a large portion of their structures including an identical 907 amu core fragment.

These data were used to develop a quantitative LC–MS assay for karlotoxins from cultures and environmental samples. The sensitivity afforded by MS detection compared to UV absorbance allowed toxin quantification at 0.2 ng when injected on column. Aqueous solutions of karlotoxins were found to quantitatively adsorb to PTFE and nylon membrane filters. Aliquots from whole cultures or environmental samples could be concentrated and desalted by adsorption to PTFE syringe filters and karlotoxins eluted with methanol for analysis by LC–MS. This simplified solid phase cleanup afforded new data indicating that each karlotoxin may also exist as sulfated derivatives and also provided a rapid detection method for karlotoxin from environmental samples and whole cultures.     

A gas chromatography–mass spectrometry method to monitor detergents removal from a membrane protein sample

In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.     

Proteomic analysis of estrogen response of premalignant human breast cells using a 2-D liquid separation/mass mapping technique

A 2-D liquid-phase separation method based on chromatofocusing and nonporous silica RP-HPLC followed by ESI-TOF-MS was used to analyze proteins in whole cell lysates from estrogen-treated and untreated premalignant, estrogen-responsive cell line MCF10AT1 cells. 2-D mass maps in the pH range 4.6-6.0 were generated with good correlation to theoretical M(r) values for intact proteins. Proteins were identified based on intact M(r), pI and PMF, or MS/MS sequencing. About 300 unique proteins were identified and 120 proteins in mass range 5-75 kDa were quantified upon treatment of estrogen. Around 40 proteins were found to be more highly expressed (>four-fold) and 17 were down-regulated (>four-fold) in treated cells. In our study, we found that many altered proteins have characteristics consistent with the development of a malignant phenotype. Some of them have a role in the ras pathway or play an important role in signal pathways. These changed proteins might be essential in the estrogen regulation mechanism. Our study highlights the use of the MCF10AT1 cell line to examine estrogen-induced changes in premalignant breast cells and the ability of the 2-D mass mapping technique to quantitatively study protein expression changes on a proteomic scale.     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.     

Doping control analysis of trenbolone and related compounds using liquid chromatography-tandem mass spectrometry

Trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one) and its derivatives such as 17α-methyltrenbolone represent a class of highly potent anabolic-androgenic steroids, which are prohibited in sports according to the regulation of the World Anti-Doping Agency (WADA). Due to marginal gas chromatographic properties of these compounds but excellent proton affinities resulting from a large and conjugated π-electron system, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the method of choice for the detection of these analytes in sports drug testing. Recent findings of trenbolone and methyltrenbolone in doping control urine samples of elite athletes demonstrated the importance of a sensitive and robust analytical method, which was based on an enzymatic hydrolysis of target compounds, liquid-liquid extraction and subsequent LC-MS/MS measurement. Diagnostic product ions obtained after collision-induced dissociation of protonated molecules were found at m/z 227, 211, 199 and 198, which enabled targeted screening using multiple reaction monitoring. Using 7 model compounds (trenbolone, epitrenbolone, methyltrenbolone, ethyltrenbolone, propyltrenbolone, 17-ketotrenbolone and altrenogest), the established method was validated for specificity, lower limits of detection (0.3-3 ng/mL), recovery (72-105%), intraday and interday precision (≤20%).     

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.     

Surface analysis of lipids by mass spectrometry: More than just imaging

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the “omics” age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.     

Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry

A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.     

Selective association of protein molecules followed by mass spectrometry.

Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo- and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near-equilibrium conditions and will be relevant to a wide range of biological systems.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.