The proteomics and interactomics of human erythrocytes.

In this minireview, we focus on advances in our knowledge of the human erythrocyte proteome and interactome that have occurred since our seminal review on the topic published in 2007. As will be explained, the number of unique proteins has grown from 751 in 2007 to 2289 as of today. We describe how proteomics and interactomics tools have been used to probe critical protein changes in disorders impacting the blood.

Functional 20S proteasomes in mature human red blood cells.

The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles.

The rat red blood cell proteome is altered by priming with 2-butoxyethanol.

Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD(90) dose (1500 mg BE/kg, p.

The isolation of reticulocyte-free human red blood cells.

We depleted reticulocytes from erythrocytes of both sickle cell disease (SCD) subjects and healthy controls by four methods: fluorescence-activated cell sorting (FACS), Miltenyi immunomagnetic depletion (MACS), a combination of these methods (FACS + MACS) and Percoll density separation. The efficiency of these methods was assessed by new methylene blue staining and manual enumeration of the reticulocytes. FACS sorted erythrocytes from reticulocytes based on size and granularity, as well as the absence of dsDNA staining.

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry.

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells.

Mouse anterior pituitary gland: analysis by ion trap mass spectrometry.

We investigated the proteome of the anterior pituitary gland (AP) in a species in which the genome has been sequenced. Subcellular fractions of APs from 2-month-old male mice were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography tandem mass spectrometry and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 g and cytosolic fractions, we identified 49, 36 and 68 different proteins, respectively.

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells.

Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its alpha subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay.

Ubiquitination of red blood cell alpha-spectrin does not affect heterodimer formation.

Erythrocyte alpha-spectrin is ubiquitinated in repeats alpha20/alpha21, which also represents the nucleation site for contact with the beta subunit which leads to heterodimer formation by a zippering mechanism. In this study we have determined the second-order rate constant for association of ubiquitinated alpha'-spectrin, nonubiquitinated alpha-spectrin, and beta-spectrin into the alpha'beta or alphabeta heterodimer. The rate constant for incorporation of monomers into heterodimers at 37 degrees C were (5.

Analysis of the golden Syrian hamster anterior pituitary gland proteome by ion trap mass spectrometry.

We utilized mass spectrometry (MS) and bioinformatics to investigate the proteome of the anterior pituitary gland (AP). Subcellular fractions of APs from 2-month-old male Golden Syrian hamsters were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography MS/MS and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 x g and cytosolic fractions we identified 76, 52 and 52 different proteins, respectively.

The human erythrocyte proteome: analysis by ion trap mass spectrometry.

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins.

Preparation of irreversibly sickled cell beta-actin from normal red blood cell beta-actin.

We have previously demonstrated that an oxidative change, the formation of a disulfide bridge between two cysteine residues, in the membrane protein beta-actin is primarily responsible for locking the irreversibly sickled red blood cells (ISCs) of sickle cell anemic patients into the sickle shape. To support studies on biological and chemical characterization of the oxidized beta-actin and pharmacological research toward the reversal of the oxidation, we attempted to prepare oxidized beta-actin from normal red blood cell (RBC) beta-actin by a chemical reaction, expecting a product equivalent to that found in ISCs. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB, or Ellman's reagent) was used for the oxidation.