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I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different treatment groups. I could either do an LCMS/MS or a Western blot. Which one would you recommend and would you do both for validation? I am looking for time- and cost-effective techniques. I'll appreciate any suggestions. Thanks!
Western blot, though is a commonly used technique and is relatively simple to do, has some issues:
- Low throughput: it is difficult to analyse multiple proteins simultaneously
- Limited cross comparability: since antibodies to different proteins can have different affinities, they cannot be compared with each other.
- Low sensitivity
- Not very quantitative
LCMS addresses all the above limitations of western blot. It is also possible to do targeted proteomics i.e. study just some few proteins instead of the entire proteome. An LCMS system has a high initial setting up cost but a relatively lower run cost. However, the mass spectrometer has to be maintained and it is not an easy job; you basically need a facility and a dedicated technical staff.
Which one would you recommend and would you do both for validation?
Which technique to use depends on what you really want to see and what resources you have at hand. It is a commonly practised approach to do a western blot for re-validating a few selected genes identified from LCMS experiment, which is just to prove that the result can be obtained using another technique too.
Although quantitative methods using MS have been developed, MS is not inherently quantitative. Quantification with MS could be quite tricky. Therefore, it is not the first choice. But, if you do not know which protein levels change and want to find proteins the expression levels of which are different between your samples you are going to compare, MS is not bad idea. In this case, validation is necessary. In other words, MS is not used to validate WB results in general.
In your case, you know which proteins you want to see. Therefore, I would suggest WB. This is a more direct way. Validation of WB may not be necessary, but it is better to get some supportive data: the protein activities in lysates, mRNA levels, etc.
The best way is to use FPLC if you know what kind of protein you're looking for.in case you don't know what are you looking for,then you can run a 2D-PAGE and after analyzing spots then use LC MS/MS to identify your proteins and then continue with FPLC ( for record FPLC is a method of HPLC or LC which is protein friendly and even you can use it to isolate and purify your chosen protein )
Technical replicate: repeated measurements of the same sample that represent independent measures of the random noise associated with protocols or equipment. 1
Technical replicates address the reproducibility of the assay or technique, but not the reproducibility of the effect or event being studied. For example, loading of replicate lanes for each sample on a blot, running replicate blots in parallel, or repeating blots with same samples on different days.
Technical replicates indicate if your measurements are scientifically robust or noisy, and how large the measured effect must be to stand out above that noise.
Figure 1. Technical replicates help identify variation in technique. For example, lysate derived from a mouse and treated under a set of experimental conditions (A, B, C), then run and measured independently three times, will help identify variation in technique.
LCMS/MS versus Western Blot - Biology
Atenolol is a β1-selective drug, which exerts greater blocking activity on β1-adrenoreceptors than on β2-adrenoreceptors, with the S-enantiomer being more active than R-enantiomer. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of atenolol using an iTRAQ-coupled two-dimensional LC-MS/MS approach. Our results indicated that some calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4, and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer, whereas metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b5 reductase, and α-N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with the S-enantiomer relative to those incubated with the R-enantiomer. The involvement of NADH-cytochrome b5 reductase in the intracellular anabolic activity was validated by NAD + /NADH assay with a higher ratio of NAD + /NADH correlating with a higher proportion of NAD + . The down-regulation of the calcium-binding proteins was possibly involved in the lower intracellular Ca 2+ concentration in A7r5 cells incubated with the S-enantiomer of atenolol. Ca 2+ signals transduced by calcium-binding proteins acted on cytoskeletal proteins such as nestin and β-tropomyosin, which can play a complex role in phenotypic modulation and regulation of the cytoskeletal modeling. Our preliminary results thus provide molecular evidence on the metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol.
Published, MCP Papers in Press, February 11, 2008, DOI 10.1074/mcp.M700485-MCP200
This work was supported in part by Academic Research Funds, Ministry of Education, Singapore (Grant RG44/06 to W. N. C.).
The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
Both authors contributed equally to this work.
Recipient of a graduate research scholarship from Nanyang Technological University.
A research assistant in the School of Chemical and Biomedical Engineering, Nanyang Technological University.
Please see our HPLC page for information on how liquid chromatography (the LC in LC-MS) is able to separate compounds. There are minor differences between normal HPLC runs and runs using LC-MS. One of these differences is that the flow rate for LC-MS is slow than the standard HPLC flow rate of 1 mL/min. In addition, conventional HPLC columns are about 100-300 mm long while the columns used for LC-MS applications are substantially smaller, typically 30-50 mm in length. LC-MS columns also have a small internal diameter which provides better separation and can function at flow rates of less than 1 mL/min.
When the molecules are eluted from the chromatography column they are under pressure and the continuous flow cannot be directly detected by the mass spectrometer because mass spec units operate in a vacuum and requires the liquid to be passed through an interface. The interface removes the mobile phase used in the chromatography step and transfers the analyte to the mass spec unit. May different types of interfaces exist but the most commonly used interfaces are electrospray ionization (ESI) systems, atmospheric pressure chemical ionization (APCI) systems, or atmospheric pressure photo-ionization (APPI) systems. In all these interfaces, the liquid is nebulized into a fine spray, ionized and then transferred to the mass spec detector.
The mass spec detector measures the mass-to-charge ratio of ions by exposing the ions to a magnetic or electric fields which can alter the movement of the ions allowing the ions to be sorted based on their mass. The detector can then measure and amplify the ion current to quantitate the amount of sorted ions. If two mass spec detectors are used, then molecules with a particular mass-to-charge ratio can be chosen to undergo further analysis by fragmenting the ion by collision-induced dissociation or other fragmentation processes. These fragmented ions can then be detected by the second mass spec unit.
The mass spec detector transfers the mass-to-charge data to a computer which graphical presents the information as a mass spectrum. The mass spectrum of the sample can be used to determine the concentration of known or unknown compounds, find the mass of impurities and give insight into chemical structures.
Can LC-MS perform quantitative analysis?
How does LC-MS perform quantitative analysis? What is the qualitative spectrum produced by mass spectrometry in liquid mass spectrometry? Since mass spectrometry is used for quantification, what is the use of the spectrum produced by the UV detector in the chromatogram itself? There are SIM and full scan for LC/MS, so which one is generally used in pharmacokinetic tests? Especially when analyzing drugs in vivo, what is the mass spectrum of internal standard method used for quantification?
Mass spectrometry quantitative analysis generally uses LC-MS/MS instead of LC-MS. Look at the mass-to-charge ratio and estimate the molecular weight. UV can be used for quantification, but when there are interference peaks of ultraviolet absorption, the quantification is not accurate. LC-MS/MS uses precursor/product ions for quantification. Relatively speaking, it has much less interference and is more accurate. It is widely used in PK/TK. Only the quantitative spectra of LC-MS/MS are here, for reference only!
The 183.1/141.3 channel is an internal standard, and the other two are compounds.
The widely accepted method of mass spectrometry quantification is MS/MS quantification. This quantification is often achieved by three-stage quadrupole or ion trap mass spectrometry. The reason why MS/MS is required is that many compounds have the same quality. When using the first dimension, single-stage mass spectrometry, MS to quantify, it also lacks specificity, especially for complex matrices like blood. The second dimension of MS (ie MS/MS) can provide the only break in most cases. Combining specific precursor ion mass and unique fragment ion information can selectively monitor quantified compounds.
What is HPLC?
High-performance liquid chromatography (HPLC) is a popular separation technique in analytical chemistry. It is mainly used to separate the components, to identify and to quantify each component in a mixture. Earlier, this method was known as high-pressure liquid chromatography because it depended on pumps to flow a pressurized liquid solvent comprising the sample mixture through a column packed with a solid adsorbent material. Each and every constituent in the sample mixture interacts differently with the solid adsorbent material, which results in different flow rates for different constituents. This can lead to the separation of the constituents as they flow out the HPLC column.
HPLC has been used for various applications such as analysis of vitamin D levels in blood, illegal drug usage of athletes by detecting the drug residues in their urine, sorting out the constituents of a complex biological sample for research purposes and analysis and manufacturing of pharmaceuticals.
A Comparative Quantitative LC-MS/MS Profiling Analysis of Human Pancreatic Adenocarcinoma, Adjacent-Normal Tissue, and Patient-Derived Tumour Xenografts
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide it develops in a relatively symptom-free manner, leading to rapid disease progression and metastasis, leading to a 5-year survival rate of less than 5%. A lack of dependable diagnostic markers and rapid development of resistance to conventional therapies are among the problems associated with management of the disease. A better understanding of pancreatic tumour biology and discovery of new potential therapeutic targets are important goals in pancreatic cancer research. This study describes the comparative quantitative LC-MS/MS proteomic analysis of the membrane-enriched proteome of 10 human pancreatic ductal adenocarcinomas, 9 matched adjacent-normal pancreas and patient-derived xenografts (PDXs) in mice (10 at F1 generation and 10 F2). Quantitative label-free LC-MS/MS data analysis identified 129 proteins upregulated, and 109 downregulated, in PDAC, compared to adjacent-normal tissue. In this study, analysing peptide MS/MS data from the xenografts, great care was taken to distinguish species-specific peptides definitively derived from human sequences, or from mice, which could not be distinguished. The human-only peptides from the PDXs are of particular value, since only human tumour cells survive, and stromal cells are replaced during engraftment in the mouse this list is, therefore, enriched in tumour-associated proteins, some of which might be potential therapeutic or diagnostic targets. Using human-specific sequences, 32 proteins were found to be upregulated, and 113 downregulated in PDX F1 tumours, compared to primary PDAC. Differential expression of CD55 between PDAC and normal pancreas, and expression across PDX generations, was confirmed by Western blotting. These data indicate the value of using PDX models in PDAC research. This study is the first comparative proteomic analysis of PDAC which employs PDX models to identify patient tumour cell-associated proteins, in an effort to find robust targets for therapeutic treatment of PDAC.
Keywords: ADC therapy PDX membrane-enriched pancreatic cancer proteomics.
Conflict of interest statement
The authors declare no conflict of interest.
Western blot analysis of ( A ) sodium potassium ATPase and ( B…
Proteomic clustering of pancreatic cancer…
Proteomic clustering of pancreatic cancer tumours and adjacent-normal tissues. ( A ) Principal…
Validation of differential expression levels…
Validation of differential expression levels for 4 candidate proteins across a larger, separate…
Species-specific immunohistochemical analysis of patient-derived…
Species-specific immunohistochemical analysis of patient-derived xenograft (PDX) tumour samples. An antibody specific for…
Proteomic clustering of pancreatic cancer…
Proteomic clustering of pancreatic cancer tumours and PDX F1 tissues. ( A )…
Expression of periplakin in a…
Expression of periplakin in a larger PDAC cohort and across multiple cancer types.…
Western blot analysis of CD55…
Western blot analysis of CD55 expression. ( A ) Expression of CD55 across…
The surfactant proteins (SPs), SP-B and SP-C, are important components of pulmonary surfactant involved in the reduction of alveolar surface tension. Quantification of SP-B and SP-C in surfactant drugs is informative for their quality control and the evaluation of their biological activity. Western blot analysis enabled the quantification of SP-B, but not SP-C, in surfactant drugs. Here, we report a new procedure involving chemical treatments and LC-MS to analyze SP-C peptides. The procedure enabled qualitative analysis of SP-C from different species with discrimination of the palmitoylation status and the artificial modifications that occur during handling and/or storage. In addition, the method can be used to estimate the total amount of SP-C in pulmonary surfactant drugs. The strategy described here might serve as a prototype to establish analytical methods for peptides that are extremely hydrophobic and behave like lipids. The new method provides an easy measurement of SP-C from various biological samples, which will help the characterization of various experimental animal models and the quality control of surfactant drugs, as well as diagnostics of human samples.
This work was supported by JSPS KAKENHI Grants 24229003 (T.S.), 25116707 (Y.K.), 23790324 (H.S.), 24790805 (T.H.), and the grants for National Center for Global Health and Medicine 24-001 and 25-201 (T.S.). Materials and fees for this research were provided, in part, by Mitsubishi Tanabe Pharma Corporation. The Department of Lipidomics, Graduate School of Medicine, University of Tokyo is financially supported by Shimadzu Corporation and Ono Pharmaceutical Company, LTD
bovine surfactant protein C
murine surfactant protein C
selected reaction monitoring
The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of two figures and one table.
Present address of T. Harayama: Department of Biochemistry, Sciences II, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
Cancer Care Centre, St George Hospital, Kogarah, 2217, NSW, Australia
Lei Chang, Jie Ni, Julia Beretov, Jingli Hao, Joseph Bucci, Peter H. Graham & Yong Li
St George and Sutherland Clinical School, Faculty of Medicine, UNSW, Kensington, 2052, NSW, Australia
Lei Chang, Jie Ni, Julia Beretov, Jingli Hao, Joseph Bucci, Peter H. Graham & Yong Li
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China
SEALS, Anatomical Pathology, St George Hospital, Kogarah, 2217, NSW, Australia
Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, UNSW, Kensington, 2052, NSW, Australia
School of Medical Science, UNSW, Kensington, 2052, NSW, Australia
Department of Urology, St George Hospital, Kogarah, 2217, NSW, Australia
David Malouf & David Gillatt
Australian School of Advanced Medicine, Macquarie University, 2109, NSW, Australia