III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphing... more III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 uM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 uM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine threeto fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.
Advances in experimental medicine and biology, 2016
Proteins may be considered as polypeptides large enough to have a well-defined tertiary, or three... more Proteins may be considered as polypeptides large enough to have a well-defined tertiary, or three-dimensional structure. In aqueous media, this structure is typically one in which polar and charged amino acid residues are on the surface while hydrophobic residues tend to be sequestered in the core and reasonably inaccessible to the aqueous environment. Proteins that are not normally found free in aqueous media, such as membrane proteins and apolipoproteins, can have tertiary structures that deviate from this model. In general, the biological activity of proteins requires the preservation of their tertiary structure, and this sets more limits upon the chromatography than is true of peptides. In proteomics, the concern is with which proteins are present and in what quantity rather than maintaining biological activity. Such applications are freer to use mobile and stationary phases that denature protein structure. However, considerations of solubility and recovery may still set more li...
We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatog... more We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatography sup-ports and a continuous, post-separation enzyme detector in a high-performance liquid chromatograph. Chromato-graphic analysis and enzyme detection are fully automated and provide excellent reproducibility. Factors affecting the isoenzyme profile and detector response characteristics are assessed. Lactate dehydrogenase and creatine kinase isoenzymes in tissue extracts, control materials used as electrophoretic standards, and serum were profiled by this method to establish the resolution and reliability of the method. We show the clinical use of this method in de-tecting changes in these isoenzymes in serum associated with acute myocardial infarction. Additional Keyphrases: creatine kinase lactate dehy-drogenase #{149} rats isoenzymeprofiling The separation of isoenzymes by ion-exchange chromatography has generally been limited to prepar-ative applications, while electrophoresis ...
ABSTRACT: One of the challenges in proteomics is the proteome’s complexity, which necessitates th... more ABSTRACT: One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromato-graphy (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard pr...
Some procedures for isolation of phosphopeptides produce samples containing nonvolatile salt that... more Some procedures for isolation of phosphopeptides produce samples containing nonvolatile salt that must be removed prior to further analysis. Solid-phase extraction (SPE) is a straightforward method for doing this. Few studies in the literature compare alternative materials for the purpose or assess the effects of salt levels on the effectiveness of the materials involved. This study does so for titania, HyperCarb® (graphitic carbon) and C-18 silica. Samples included a synthetic peptide with 1-4 phosphates, the tryptic digest of beta-casein, and two fractions from a tryptic digest of HeLa cell lysate: a low-salt fraction rich in singly phosphorylated peptides and a high-salt fraction enriched in peptides with more than one phosphate. All SPE materials were packed into TopTips™ or Sep-Pak® cartridges. Filtrates, washes and eluates were analyzed via ERLIC (Electrostatic Repulsion-Hydrophilic Interaction Chromatography), a new mode with high selectivity and resolution for tryptic phosph...
A well-hydrated counterion can selectively and dramatically increase retention of a charged analy... more A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography. The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity ranging from 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, and N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with "crosstalk" modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase liquid chromatography-tandem mass spectrometry analysis. Between 47-100% of the peptides with more than one phosphate or sialyl residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.
Although top-down proteomics has emerged as a powerful strategy to characterize proteins in biolo... more Although top-down proteomics has emerged as a powerful strategy to characterize proteins in biological systems, the analysis of endogenous membrane proteins remains challenging due to their low solubility, low abundance, and the complexity of the membrane subproteome. Here, we report a simple but effective enrichment and separation strategy for top-down proteomics of endogenous membrane proteins enabled by cloud point extraction and multidimensional liquid chromatography coupled to high-resolution mass spectrometry (MS). The cloud point extraction efficiently enriched membrane proteins using a single extraction, eliminating the need for time-consuming ultracentrifugation steps. Subsequently, size-exclusion chromatography (SEC) with an MS-compatible mobile phase (59% water, 40% isopropanol, 1% formic acid) was used to remove the residual surfactant and fractionate intact proteins (6-115 kDa). The fractions were separated further by reversed-phase liquid chromatography (RPLC) coupled with MS for protein characterization. This method was applied to human embryonic kidney cells and cardiac tissue lysates to enable the identification of 188 and 124 endogenous integral membrane proteins, respectively, some with as many as 19 transmembrane domains.
Middle-down proteomics has emerged as the method of choice to study combinatorial histone post tr... more Middle-down proteomics has emerged as the method of choice to study combinatorial histone post translational modifications (PTMs). In the common bottom-up workflow, histones are digested into relatively short peptides (4-20 aa), separated using reversed-phase chromatography and analyzed using typical proteomics methods in mass spectrometry. In middle-down, histones are cleaved into longer polypeptides (50-60 aa) mostly corresponding to their N-terminal tails, resolved using weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) and analyzed with less conventional mass spectrometry, i.e. using Electron Transfer Dissociation (ETD) for analyte fragmentation. Middle-down is not nearly as utilized as bottom-up for PTM analysis, partially due to its limited reproducibility and robustness. This has also limited the establishment of rigorous benchmarks to discriminate good vs poor quality experiments. Here, we describe critical aspects of the middle-down workflow to assist the user in evaluating the presence of biased and misleading results. Specifically, we tested the use of porous graphitic carbon (PGC) during the desalting step, demonstrating that desalting using only C18 material leads to sample loss. We also tested different salts in the WCX-HILIC buffers for their effect on retention, selectivity, and reproducibility of analysis of variants of histone tail fragments, in particular replacing ammonium ion with ethylenediammonium ion in buffer A. These substitutions had marked effects on selectivity and retention. Our results provide a streamlined way to evaluate middle-down performance to identify and quantify combinatorial histone PTMs.
We describe a rapid, simple, and sensitive high-performance liquid chromatographic method for the... more We describe a rapid, simple, and sensitive high-performance liquid chromatographic method for the determination of hemoglobin A1c with a bonded-phase cation-exchange column. About five measurements are obtained per hour on small quantities of whole blood. Sample preparation requires no centrifugation or washing of cells. The results correlate well (r = 0.918, n = 51) with a commercially available, disposable mini-column kit. The results are insensitive to small changes in pH and temperature. Variants arising from hemoglobinopathies display different chromatographic profiles.
If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can b... more If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase. This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained. This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides. ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means. In addition, phosphopeptides can be isolated selectively from a tryptic digest.
Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify pro... more Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.
Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analy... more Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules, but typically suffers from low resolution. Herein we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10-223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that 2D sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to...
III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphing... more III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 uM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 uM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine threeto fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.
Advances in experimental medicine and biology, 2016
Proteins may be considered as polypeptides large enough to have a well-defined tertiary, or three... more Proteins may be considered as polypeptides large enough to have a well-defined tertiary, or three-dimensional structure. In aqueous media, this structure is typically one in which polar and charged amino acid residues are on the surface while hydrophobic residues tend to be sequestered in the core and reasonably inaccessible to the aqueous environment. Proteins that are not normally found free in aqueous media, such as membrane proteins and apolipoproteins, can have tertiary structures that deviate from this model. In general, the biological activity of proteins requires the preservation of their tertiary structure, and this sets more limits upon the chromatography than is true of peptides. In proteomics, the concern is with which proteins are present and in what quantity rather than maintaining biological activity. Such applications are freer to use mobile and stationary phases that denature protein structure. However, considerations of solubility and recovery may still set more li...
We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatog... more We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatography sup-ports and a continuous, post-separation enzyme detector in a high-performance liquid chromatograph. Chromato-graphic analysis and enzyme detection are fully automated and provide excellent reproducibility. Factors affecting the isoenzyme profile and detector response characteristics are assessed. Lactate dehydrogenase and creatine kinase isoenzymes in tissue extracts, control materials used as electrophoretic standards, and serum were profiled by this method to establish the resolution and reliability of the method. We show the clinical use of this method in de-tecting changes in these isoenzymes in serum associated with acute myocardial infarction. Additional Keyphrases: creatine kinase lactate dehy-drogenase #{149} rats isoenzymeprofiling The separation of isoenzymes by ion-exchange chromatography has generally been limited to prepar-ative applications, while electrophoresis ...
ABSTRACT: One of the challenges in proteomics is the proteome’s complexity, which necessitates th... more ABSTRACT: One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromato-graphy (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard pr...
Some procedures for isolation of phosphopeptides produce samples containing nonvolatile salt that... more Some procedures for isolation of phosphopeptides produce samples containing nonvolatile salt that must be removed prior to further analysis. Solid-phase extraction (SPE) is a straightforward method for doing this. Few studies in the literature compare alternative materials for the purpose or assess the effects of salt levels on the effectiveness of the materials involved. This study does so for titania, HyperCarb® (graphitic carbon) and C-18 silica. Samples included a synthetic peptide with 1-4 phosphates, the tryptic digest of beta-casein, and two fractions from a tryptic digest of HeLa cell lysate: a low-salt fraction rich in singly phosphorylated peptides and a high-salt fraction enriched in peptides with more than one phosphate. All SPE materials were packed into TopTips™ or Sep-Pak® cartridges. Filtrates, washes and eluates were analyzed via ERLIC (Electrostatic Repulsion-Hydrophilic Interaction Chromatography), a new mode with high selectivity and resolution for tryptic phosph...
A well-hydrated counterion can selectively and dramatically increase retention of a charged analy... more A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography. The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity ranging from 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, and N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with "crosstalk" modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase liquid chromatography-tandem mass spectrometry analysis. Between 47-100% of the peptides with more than one phosphate or sialyl residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.
Although top-down proteomics has emerged as a powerful strategy to characterize proteins in biolo... more Although top-down proteomics has emerged as a powerful strategy to characterize proteins in biological systems, the analysis of endogenous membrane proteins remains challenging due to their low solubility, low abundance, and the complexity of the membrane subproteome. Here, we report a simple but effective enrichment and separation strategy for top-down proteomics of endogenous membrane proteins enabled by cloud point extraction and multidimensional liquid chromatography coupled to high-resolution mass spectrometry (MS). The cloud point extraction efficiently enriched membrane proteins using a single extraction, eliminating the need for time-consuming ultracentrifugation steps. Subsequently, size-exclusion chromatography (SEC) with an MS-compatible mobile phase (59% water, 40% isopropanol, 1% formic acid) was used to remove the residual surfactant and fractionate intact proteins (6-115 kDa). The fractions were separated further by reversed-phase liquid chromatography (RPLC) coupled with MS for protein characterization. This method was applied to human embryonic kidney cells and cardiac tissue lysates to enable the identification of 188 and 124 endogenous integral membrane proteins, respectively, some with as many as 19 transmembrane domains.
Middle-down proteomics has emerged as the method of choice to study combinatorial histone post tr... more Middle-down proteomics has emerged as the method of choice to study combinatorial histone post translational modifications (PTMs). In the common bottom-up workflow, histones are digested into relatively short peptides (4-20 aa), separated using reversed-phase chromatography and analyzed using typical proteomics methods in mass spectrometry. In middle-down, histones are cleaved into longer polypeptides (50-60 aa) mostly corresponding to their N-terminal tails, resolved using weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) and analyzed with less conventional mass spectrometry, i.e. using Electron Transfer Dissociation (ETD) for analyte fragmentation. Middle-down is not nearly as utilized as bottom-up for PTM analysis, partially due to its limited reproducibility and robustness. This has also limited the establishment of rigorous benchmarks to discriminate good vs poor quality experiments. Here, we describe critical aspects of the middle-down workflow to assist the user in evaluating the presence of biased and misleading results. Specifically, we tested the use of porous graphitic carbon (PGC) during the desalting step, demonstrating that desalting using only C18 material leads to sample loss. We also tested different salts in the WCX-HILIC buffers for their effect on retention, selectivity, and reproducibility of analysis of variants of histone tail fragments, in particular replacing ammonium ion with ethylenediammonium ion in buffer A. These substitutions had marked effects on selectivity and retention. Our results provide a streamlined way to evaluate middle-down performance to identify and quantify combinatorial histone PTMs.
We describe a rapid, simple, and sensitive high-performance liquid chromatographic method for the... more We describe a rapid, simple, and sensitive high-performance liquid chromatographic method for the determination of hemoglobin A1c with a bonded-phase cation-exchange column. About five measurements are obtained per hour on small quantities of whole blood. Sample preparation requires no centrifugation or washing of cells. The results correlate well (r = 0.918, n = 51) with a commercially available, disposable mini-column kit. The results are insensitive to small changes in pH and temperature. Variants arising from hemoglobinopathies display different chromatographic profiles.
If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can b... more If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase. This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained. This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides. ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means. In addition, phosphopeptides can be isolated selectively from a tryptic digest.
Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify pro... more Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.
Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analy... more Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules, but typically suffers from low resolution. Herein we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10-223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that 2D sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to...
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Papers by Andrew Alpert