Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric
methods based on stable isotope quantitation have shown great promise ...for the simultaneous and automated identification and
quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for s table i sotope l abeling by a mino acids in c ell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard
essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated
leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced
by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings
in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after
harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either
all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression
during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include
glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate
procedure that can be used as a quantitative proteomic approach in any cell culture system.
A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the
discovery of a novel protein named STAP ( st ellate cell a ...ctivation-associated p rotein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid
sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoing in vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such
as smooth muscle α-actin, PDGF receptor-β, and neural cell adhesion molecule. The expression of STAP protein and mRNA was
augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected
STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed
that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These
results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both
of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP
could thus play a role as an antifibrotic scavenger of peroxides in the liver.
Proteome analysis was performed on cellular and secreted proteins of normal (quiescent) and activated rat hepatic stellate cells. The stellate cells were activated either in vitro by cultivating ...quiescent stellate cells for 9 days or in vivo by injecting rats with carbon tetrachloride for 8 weeks. A total of 43 proteins/polypeptides were identified, which altered their expression levels when the cells were activated in vivo and/orin vitro . Twenty-seven of them showed similar changesin vivo and in vitro, including up-regulated proteins such as calcyclin, calgizzarin, and galectin-1 as well as down-regulated proteins such as liver carboxylesterase 10 and serine protease inhibitor 3. Sixteen of them showed different expression levels between in vivo and in vitro activated stellate cells. These results were reproducibly obtained in 3 independent experiments. The up-regulation of calcyclin, calgizzarin, and galectin-1, as well as the down-regulation of liver carboxylesterase 10 were directly confirmed in fibrotic liver tissues. Northern blots confirmed up-regulation of the messenger RNAs (mRNAs) of calcyclin, calgizzarin, and galectin-1 in activated stellate cells, indicating that these changes were controlled at the mRNA level. In addition a list compiling over 150 stellate cell proteins is presented. The data presented here thus provide a significant new protein-level insight into the activation of hepatic stellate cells, a key event in liver fibrogenesis. (Hepatology 2000;32:268-277.)
Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic ...digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.
Graphical abstract
This study developed an enzymatic method for high‐throughput mapping of phosphoproteins on two‐dimensional (2‐D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two ...aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2‐D electrophoresis. Phosphoproteins could be mapped on the 2‐D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine‐rich tetratricopeptide repeat‐containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high‐throughput mapping of phosphoproteins in proteome research.
This study demonstrates structural and conformational characterization of proteins by nanoflow electrospray ionization (nanoESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) utilizing ...a quadrupole time‐of‐flight (Q‐TOF) mass spectrometer (Micromass, Manchester, England). Model peptides were successfully sequenced at the 35 attomole (amol) level, and peptides derived from a tryptic in‐gel digest of 25 femtomole (fmol) bovine serum albumin (BSA) were successfully sequenced. The results demonstrated that the MS/MS sensitivity of the Q‐TOF clearly surpassed the detection limit of the silver stain. A silver destaining step greatly improved the mass analysis of peptides derived from in‐gel digests. Interestingly, sequence analysis revealed BSA residue 424 (tyrosine) as a potential chlorination site. In addition, a modified procedure was successfully used to extract and measure the masses of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE)‐resolved proteins in the 10—68.5 kDa range. The Q‐TOF was also used to monitor conformational changes of proteins. These experiments demonstrated an acid‐induced denaturation of BSA in the pH 3—4 range, and heat‐induced unfolding of cytochrome c between 50 and 60°C. Finally, Zn2+ binding was demonstrated for the carbonic anhydrase apoprotein. In summary, the wide range of applications and the high quality of the experimental data made the Q‐TOF mass spectrometer a powerful analytical tool for protein characterization.
The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog,
Rana catesbeiana, was investigated utilizing newly cloned
Rana keratin cDNAs as probes.
Rana larval ...keratin (RLK) cDNA (
rlk) was cloned using highly specific antisera against
Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The
Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned
rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of
rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (
rak) of
Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a
Rana keratin 8 (RK8) cDNA (
rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and
in situ hybridization experiments showed that
rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of
rlk and
rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.
Human 2-D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi-bin/celis). The databanks, which offer a ...comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2-D PAGE reference maps (non-cultured keratinocytes, non-cultured transitional cell carcinomas, MRC-5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2-D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss-Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on-line information is updated regularly.
This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells ...including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.
LC MS/MS has become an established technology in proteomic studies, and with the maturation of the technology the bottleneck
has shifted from data generation to data validation and mining. To address ...this bottleneck we developed Experimental Peptide
Identification Repository (EPIR), which is an integrated software platform for storage, validation, and mining of LC MS/MS-derived
peptide evidence. EPIR is a cumulative data repository where precursor ions are linked to peptide assignments and protein
associations returned by a search engine ( e.g. Mascot, Sequest, or PepSea). Any number of datasets can be parsed into EPIR and subsequently validated and mined using a
set of software modules that overlay the database. These include a peptide validation module, a protein grouping module, a
generic module for extracting quantitative data, a comparative module, and additional modules for extracting statistical information.
In the present study, the utility of EPIR and associated software tools is demonstrated on LC MS/MS data derived from a set
of model proteins and complex protein mixtures derived from MCF-7 breast cancer cells. Emphasis is placed on the key strengths
of EPIR, including the ability to validate and mine multiple combined datasets, and presentation of protein-level evidence
in concise, nonredundant protein groups that are based on shared peptide evidence.
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