Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC–ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and ¹³C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography–electrospray ionization and mass spectrometry. Four independent methods, (i) internal ¹³C isotope PA dilution curves, (ii) internal ¹³C isotope one-point calibration, (iii) external PA standard curve, and (iv) external ¹³C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

<div><p>The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute regulation by treatment with A-769662, at least some of which is mediated by the metabolic sensor AMPK.</p></div

Inhibition of AMPK by siRNA gene silencing or by compound C prevents the reduction in cell surface β1-integrin elicited by A-769662 treatment.

<p>(<b><i>A-C)</i></b> RPE cells were transfected with siRNA targeting AMPK α1/2 or non-targeting (NT, control) siRNA. (<b><i>A</i></b>) Whole cell lysates were prepared and resolved by immunoblotting and probed with anti-AMPK α1/2 or anti-actin antibodies. Shown are immunoblots representative of at least 3 independent experiments. (<b><i>B</i></b>) Following siRNA transfection, cells were treated with 100 μM A-769662 for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>C)</i></b> Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments). (<b><i>D</i></b>) RPE cells were treated with 100 μM A-769662 or 40 μM compound C, alone or in combination, for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>E)</i></b> Cell surface β1-integrin levels obtained by fluorescence microscopy as in (D) were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments).</p

Proteins with cell adhesion and migration GO classification depleted from the cell surface upon A-769662 treatment.

<p>Shown are the 32 proteins with Cell Adhesion and Migration GO classification detected in the cell surface fraction of control but not A-769662 treated cells. Shown for each are the detected peptide counts in each of the treatment conditions.</p><p>Proteins with cell adhesion and migration GO classification depleted from the cell surface upon A-769662 treatment.</p

AMPK activation and mass spectrometry analysis of cell surface proteins.

<p>(<b><i>A</i></b>) RPE cells were stimulated with 100 μM A-769662 in media containing 0.1% FBS for indicated times. Shown are representative immunoblots using antibodies as indicated. (<b><i>B</i></b>) Shown is a diagram depicting cell stimulation, surface biotinylation, purification of biotinylated proteins, mass spectrometry and peptide identification. We thus identified a total of 838 proteins within all cell surface fractions, of which 653 exhibited reduced detection in the cell surface fraction of cells treated with A-769662, 93 proteins exhibited increased cell surface abundance in cells treated with A-769662, and a further 92 were classified as exhibiting largely unaltered detection in the cell surface fraction upon AMPK activation. A complete list of identified proteins can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128013#pone.0128013.s001" target="_blank">S1 Table</a>.</p

Treatment with A-769662 reduces cell surface β1-integrin levels.

<p>(<b><i>A</i></b>) RPE cells were stimulated with 100 μM A-769662 for 90 min or left unstimulated (basal). Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>B</i></b>) Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified as described in <i>Materials and Methods</i> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128013#pone.0128013.s009" target="_blank">S5B Fig</a>. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 4 independent experiments). (<b><i>C</i></b>) RPE cells were stimulated with 2 mM AICAR for 90 min or left unstimulated (basal), followed by cell-surface biotinylation, purification of biotinylated proteins and immunoblotting of fractions with an antibody specific to β1-integrin. Shown is an immunoblot of cell surface β1-intergin (<i>top panel</i>, corresponding to the streptavidin pull-down), and of the corresponding intracellular β1-integrin (<i>bottom panel</i>, corresponding to the above supernatant), representative of 4 independent experiments. (<b><i>D</i></b>) Shown are representative immunoblots of whole-cell lysates prepared from cells stimulated with either 100 μM A-769662, 2 mM AICAR, 40 μM compound C (each for 90 min) or left unstimulated (control), probed with antibodies to detect total cellular β1-integrin or actin (load).</p