Acrylamide--a cysteine alkylating reagent for quantitative proteomics.

Mass spectrometry-based relative quantification of proteins is often achieved by the labeling of two samples with isotopically light and heavy reagents. The intensities of the ions with different masses, but same chemical properties, can be reliably used for determining relative quantities. Several strategies of labeling with various weakness and strength and degrees of complexity have been described. In this chapter, we describe a simple and inexpensive protein-labeling procedure based on the use of acrylamide and deuterated acrylamide as a cysteine alkylating reagent. Gel electrophoresis is one of the most commonly used techniques for analyzing/visualizing proteins, thus, we emphasize the use of acrylamide as a labeling procedure for quantifying proteins isolated by one- and two-dimensional polyacrylamide gel electrophoresis.

The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

ATRX syndrome is characterized by X-linked mental retardation associated with alpha-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.

Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIalpha and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner.

The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis.

Gel electrophoresis is often used for the primary analysis and purification of proteins, and peptide mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for the rapid identification of unknown proteins. The identification is usually obtained by digesting the protein with an enzyme and matching the masses of the proteolytic peptides with those of each protein in a sequence database. Another important aspect in many proteomic experiments is the determination of the relative protein quantities (e.g. comparison between control and altered states). Usually, this is obtained by comparing the spot intensities of two independent gels. This procedure is time-consuming and not very accurate. Recently, several methodologies using isotope labeling of proteins for quantitative proteomic studies have been introduced (e.g. using ICAT reagents or growing cells in isotopically enriched nutrients). However, none of these methodologies is foolproof and there is still the need for simple and inexpensive alternatives for determining the relative quantities of proteins. Previously, we showed that a mixture of acrylamide and deuterated acrylamide could be used as cysteine alkylating reagent prior to electrophoresis, improving the coverage and the confidence of the protein identification procedure (Sechi S, Chait BT. Anal. Chem. 1998; 70: 5150). Here we show that a similar approach can be used to obtain relative quantitation at the femtomole level of proteins isolated by gel electrophoresis. Deuterated acrylamide is used to alkylate the cysteines in one sample and regular acrylamide is used to alkylate the cysteines in the second sample. The two samples are then mixed together in a 1:1 ratio and the relative protein quantities are determined from the ion intensity ratios of the two cysteine-containing peptides isotopic envelopes (regular/deuterated). The analysis of several proteins mixed in different ratios is reported showing that this approach can reliably be used for protein identification and quantification. Briefly, a simple and inexpensive method for quantifying and simultaneously identifying proteins isolated by gel electrophoresis using MALDI-MS is presented.