Peter Mose Larsen

2D or not 2D

Abstract 2D gel electrophoresis is the technology that everyone loves to hate—it requires manual dexterity and precision to reproduce precisely and is thus not well-suited as a high-throughput technology. Although almost everyone would like to replace it, the resolution and sensitivity it offers are exquisite and unsurpassed if one wants a global view of cellular activity. There have been several recent developments, for example, the detection of low abundance proteins, and the resolution possible with narrow-range IPG gels.

Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation

Abstract A 36K MW polypeptide was previously identified by two-dimensional gel electrophoresis whose relative proportion increases in S phase of HeLa cells, and following viral transformation of mouse 3T3B and hamster BHK21 cells. We report here experiments that show that this polypeptide (isoelectric focusing (IEF 49)) is localized in the nucleus and that its relative proportion decreases dramatically under conditions for which there is a decrease in the rate of cell proliferation, due for example, to the formation of giant HeLa cells by X-ray irradiation or the senescence of human skin fibroblasts. It is present in negligible amounts in non-cycling cells of adult tissues. These studies also revealed and unrelated cytoplasmic polypeptide (IEF 52; tropomyosin related; coordinates 35/1.43) whose relative proportion increased significantly and reproducibly with a decrease in the rate of cell proliferation. It is suggested that IEF 49 may be a useful marker for cycling cells.

Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients.

The rising number of proteome projects leads to new challenges for two‐dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4—12 or 3—12 (Görg et al., Electrophoresis 1999, 20, 712—717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer‐aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4—5, 4.5—5.5, 5—6, 5.5—6.7 and 6—9 are presented and compared to the pH 4—7 and 3—10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3—10 gradient.

Phospho-proteomics: Evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis

Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de‐phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in‐gel digested phospho‐proteins. Phospho‐peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho‐peptides requires more protein than normally available in our proteomics studies.

Architecture and polypeptide composition of HeLa cytoskeletons: Modification of cytoarchitectural polypeptides during mitosis

Abstract Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin). Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 M r region as determined by staining with Coomassie blue and labelling with a mixture of 14 C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF ‡ polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 M rmr ) and six polypeptides (IEF 12, 68,000 M r ; IEF 24, 56,000 M r ; IEF 31, 50,000 M r ; IEF 35, 49,000 M r ; IEF 36, 48,500 M r and IEF 46, 43,500 M r ) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis. Labelling of mitotic and interphase cells with [ 35 S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.

Comparison of yeast cell protein solubilization procedures for two‐dimensional electrophoresis

Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two‐dimensional (2‐D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with “standard” lysis buffer (9 M urea, 2% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS)), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)‐HCl, pH 7.0, followed by dilution with “standard” lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea / 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre‐boiling with SDS and using thiourea/urea lysis buffer instead of “standard” lysis buffer (procedure iii).

Coexistence of three major isoactins in a single sarcoma 180 cell

Actin is transformed sarcoma 180 cells is composed of the nonmuscle beta and gamma species and of a third, more acidic stable variant termed zeta. Two-dimensional peptide analysis shows that zeta is similar to beta actin, differing in the mobility of only one tryptic peptide. Several lines of evidence indicate that zeta is not a modified beta-actin species. This third actin species comprises 20% of the total labeled actin, has the same molecular weight as the beta and gamma actins and has a different mobility in isoelectric focusing gels from that of the known alpha actins from skeletal, cardiac and vascular smooth muscle. Like beta and gamma actin, zeta can be extracted with the actin depolymerizing factor from slime mold. Two-dimensional gel electrophoresis (isoelectric focusing) of the 35S-methionine-labeled polypeptides synthesized by a single sarcoma 180 cell showed that all three major actin species coexist within the same cell. This analysis also showed for the first time the coexistence and alpha and beta tubulin, vimentin, alpha actinin and three other polypeptides present in intermediate-filament-enriched cytoplast cytoskeletons (spots 12, 24 and 31). Determination of the ratio of gamma plus beta to zeta actin in different cytoskeletal preparations of intact and enucleated sarcoma 180 cells indicated that this actin species is not localized specifically to any of the major actin-containing structures preserved in the cytoskeletons.

Revelation of Specificity of 64K Autoantibodies in IDDM Serums by High-Resolution 2-D Gel Electrophoresis: Unambiguous Identification of 64K Target Antigen

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of β-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated α and β, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and > 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Analyses of differential proteome of human synovial fibroblasts obtained from arthritis

There is mounting evidence indicating that the synovial fibroblasts (SFs) contribute to the pathogenesis of rheumatoid arthritis (RA). The present study showed the differential proteins expression pattern of SFs from patients with RA or osteoarthritis (OA) and healthy control. Cellular proteins of cultured SFs were subjected to 2-DE and visualized by silver nitrate staining. A total of 49 spots that were statistically and differentially overexpressed in RA or OA in comparison to healthy ones were identified by MALDI-TOF-MS, and 25 proteins were successfully identified. Western blot was used to further verify some of the differential proteins. These proteins included enzymatic and structural proteins, signal transduction proteins, calcium binding protein, etc. From all of the identified proteins, a number of proteins have been implicated that involved in the healthy or pathological SFs function (e.g., S100A4, S100A10, cathepsin D) or that have potential diagnostic and prognostic value for RA (α-enolase and TPI) or that may be the new therapeutic targets (Annexin, SOD, PRX).

Comparison of the Proteomes of Three Yeast Wild Type Strains: CEN.PK2, FY1679 and W303

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains. To characterise these differences we have compared the protein expression levels between CEN.PK2, FY1679 and W303 strains using twodimensional gel electrophoresis and identified selected proteins by mass spectrometric analysis. We have found that FY1679 and W303 strains are more similar to each other than to the CEN.PK2 strain. This study identifies 62 proteins that are differentially expressed between the strains and provides a valuable source of data for the interpretation of yeast mutant phenotypes observed in CEN.PK2, FY1679 and W303 strains.