MOESM1 of Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry

Additional file 1. DHP pass 17 distinct protein matrix

MOESM1 of Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry

Additional file 1. DHP pass 17 distinct protein matrix

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

MOESM1 of Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Additional file 1: Fig. S1. A replicate CBBR stained gel to confirm the equal loading of the Western blot shown in Figure 8 of the main paper. (see legend of Figure 8 for details)

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