| 1. |
- Boström, Tove, et al.
(författare)
-
Antibodies as means for selective mass spectrometry
- 2016
-
Ingår i: Journal of chromatography. B. - : Elsevier. - 1570-0232 .- 1873-376X. ; 1021, s. 3-13
-
Tidskriftsartikel (refereegranskat)abstract
- For protein analysis of biological samples, two major strategies are used today; mass spectrometry (MS) and antibody-based methods. Each strategy offers advantages and drawbacks. However, combining the two using an immunoenrichment step with MS analysis brings together the benefits of each method resulting in increased sensitivity, faster analysis and possibility of higher degrees of multiplexing. The immunoenrichment can be performed either on protein or peptide level and quantification standards can be added in order to enable determination of the absolute protein concentration in the sample. The combination of immunoenrichment and MS holds great promise for the future in both proteomics and clinical diagnostics. This review describes different setups of immunoenrichment coupled to mass spectrometry and how these can be utilized in various applications.
|
|
| 2. |
- Boström, Tove, et al.
(författare)
-
Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomics
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- An important topic of discussion in proteomics is the degree of correlation of RNA and protein levels in cells, tissues and organs. In this study, the difference in protein and mRNA levels for a number of selected gene targets were investigated across six human cell lines using quantitative proteomics and next generation sequencing-based transcriptomics. The copy numbers of 32 proteins were determined using an absolute quantitative proteomics approach (PrEST-SILAC), where heavy isotope-labeled protein fragments were used as internal standards. A cross evaluation of protein copy numbers determined by mass spectrometry and staining profiles using immunohistochemistry showed good correlation. The mRNA levels were determined using RNA sequencing based on digital counting of sequencing reads and the levels determined as FPKM values. Comparison of the relative variations in mRNA and protein levels for individual genes across the six cell lines showed correlation between protein and mRNA levels, including six genes with high variability in expression levels in the six cell lines resulting in an average correlation of 0.9 (Spearman's rank coefficient). In summary, the analysis of the selected protein targets supports the conclusion that the translation rate across cell lines correlates for a particular gene, suggesting that individual protein levels can be predicted from the respective mRNA levels by defining the relation between protein and mRNA, specific for each human gene.
|
|
| 3. |
- Colwill, Karen, et al.
(författare)
-
A roadmap to generate renewable protein binders to the human proteome
- 2011
-
Ingår i: Nature Methods. - : Nature America Inc.. - 1548-7091 .- 1548-7105. ; 8:7, s. 551-8
-
Tidskriftsartikel (refereegranskat)abstract
- Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.
|
|
| 4. |
- Mikkonen, Saara, 1987-
(författare)
-
Sample preconcentration in open microchannels : Combinations with MALDI and nano-ESI mass spectrometry and computer simulations
- 2014
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis a novel concept for preconcentration of biomolecules in open microchannels is presented. The preconcentration is based on electromigration of charged analytes, and detection is performed with matrix-assisted laser desorption/ionization (MALDI) or nano-electrospray ionization (nESI) mass spectrometry (MS).Analysis of minute volumes of low-concentration samples is an important and challenging task within several fields of chemistry, biology and medicine. In bioanalytical chemistry in particular, sample pretreatment procedures including preconcentration must frequently be applied. Due to the often small available sample volumes, it is advantageous to perform these pretreatments in microfluidic devices. Moreover, since MS in many cases is the detection method of choice, there is a requirement for developing suitable interfacing techniques between the microchip and MS.In Paper I, the preconcentration concept is presented; silicon microchips with parallel open channels were used. The channels have a rectangular shape and are 1 cm long, 50-150 µm wide and 50 µm deep, yielding a total channel volume of 25-75 nL. By supplying sample to the channel and applying a voltage over the channel length, charged analytes will migrate towards the oppositely charged electrode and become concentrated. In Paper I, detection was performed by using the open microchannel directly as a MALDI-target. To achieve this, matrix solution was added to the channel after the preconcentration with electrospray matrix deposition. Using this approach, preconcentration of cytochrome c was achieved, and the lowest initial protein concentration successfully detected after preconcentration was 1 nM. The trypsin digest of cytochrome c was also analyzed, and the peptides were preconcentrated at different ends of the channel based on charge.Other means of coupling the preconcentration to MS, by extracting a nanovolume of the preconcentrated sample from the open channel, are presented in Paper II. The extracted samples could either be analyzed directly using nESI- or MALDI-MS, or subjected to further pretreatment (such as enzymatic digestion) in a nanodroplet under a fluorocarbon (FluorinertPTMP) liquid lid prior to MS-analysis. Furthermore, in Paper II the method was applied on an amyloid beta cell culture. This resulted in that peptides not detectable without preconcentration easily could be detected with MALDI-MS in nanodroplets extracted from the microchannels after preconcentration.Paper III includes theoretical simulations of the preconcentration procedure obtained using the electrophoresis simulator GENTRANS. The experimental results from Paper I are compared to simulations of similar systems, and simulations of an isoelectric focusing (IEF) procedure for proteins or peptides in a mixture of amino acids, are presented. The IEF procedure is to be used in the open microchannels in future experimental work.
|
|
| 5. |
- Tegel, Hanna, et al.
(författare)
-
High throughput generation of a resource of the human secretome in mammalian cells
- 2020
-
Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 58, s. 45-54
-
Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic beta-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.
|
|
| 6. |
|
|
| 7. |
- Uhlén, Mathias, et al.
(författare)
-
Tissue-based map of the human proteome
- 2015
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6220, s. 1260419-
-
Tidskriftsartikel (refereegranskat)abstract
- Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
|
|
| 8. |
- Älgenäs, Cajsa, et al.
(författare)
-
Antibody performance in western blot applications is context- dependent
- 2014
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 9:3, s. 435-445
-
Tidskriftsartikel (refereegranskat)abstract
- An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.
|
|
