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Why should I use Proteomics?

-The proteome provides 50-fold more cellular information than the genome: there are ~23,000 human genes, ~100,000 transcripts, and 20 million proteins.
-Proteins are the molecules that conduct the cell’s business. The genes of every cell in your body are identical, but the proteome of a neuron doesn’t resemble that of an adipocyte. -Because of transcriptional and translational levels of cellular control, not every gene is transcribed, and not every RNA is translated. Conversely, stable proteins often outlive transcripts from which they were made.

-Proteins are preferred targets for therapeutic agents and diagnostic tests. Immunoassays are quick, easy, capable for point-of-care use, and cost effective.
-20% of mice respond to anti-PDL1 immunotherapy. They were ISOGENIC!

Why should I care about post translational modification (PTM)?

-PTM affects structure, stability, folding, function, activity, localization, and protein-protein interactions. Basically, it controls everything that defines proteins as proteins. -Malfunction in PTM directly results in diseases like cancer, heart disease and neurodegeneration.

-Almost 10% of the genome is dedicated to the machinery that controls just two forms of PTM, ubiquitylation and phosphorylation. There are ~200 forms total.
-Protein modifications facilitate the rapid and dynamic cellular responses to stimuli, both intra- and extracellular in origin.

-Specifically and differently modified forms of protein (“protoeforms”) are what afford the mechanical complexity necessary within human cells. Modified proteins represent the ‘true’ proteome (or meta-proteome) of a given cell.
-The Central Dogma of molecular biology was articulated as phosphate signaling was being revealed and ~20 years before the discovery of the proteasome. Protein maturation is the unstated fourth step, and degradation the unstated fifth: both processes are driven by PTM.

What is your capabilities and instrumentation for performing traditional proteomics (i.e., component analysis)?
We utilize two Thermo Fisher Hybrid LTQ-OrbitrapXL Mass Spectrometers with nano- capillary HPLC, Applied Biosystems 5500 Q Trap with HPLC, Applied Biosystems Voyager- DE PRO MALDI TOF spectrometer, or the latest in orbitrap technology currently available- the Thermo Fisher Q Exactive Plus and Q Exactive HF spectrometers. We can utilize standard databases or create one customized to your needs. These machines excel in targeted proteomics, such as characterizing the phosphorylation status of a particular protein, but occasionally we use them for discovery approaches too.

To characterize known proteins for interaction with drugs, antibodies, and/or other proteins, we utilize a fully-automated BI-4500 5-Channel surface plasmon resonance (SPR) system (Biosensing Instrument) with Bi-DirectFlowTM technology, ideal for ultra-fast kinetics which produces superior data at an affordable price. Material needed is only 50 μg of protein(s), concentration, and molecular weight(s).

How do these techniques compare with Snapshot Proteomics?

The following three questions will provide additional insight, but broadly speaking traditional approaches provide a sense of what components were in the complex sample (lysate or serum commonly) while Snapshot Proteomics provides information about the activities therein. Owing to a lack of capable assay, the field has until now limited itself to consideration of the components. But even structural proteins exist for the purpose of facilitating cellular activities.

How is Snapshot Proteomics superior to mass spectrometry?

Tandem Mass Spectrometry (MS/MS) is a technique that has driven many discoveries in proteomics. However, MS/MS has two important limitations: inherently poor reproducibility and the inability to feasibly detect novel PTM*. These issues are particularly problematic for clinical applications like biomarker discovery and drug development.

-Reproducibility from biological duplicates is ~60% for MS/MS, ≥95% for Snapshot Proteomics.
-The best MS/MS runs return information on a few thousand of the most abundant proteins. Each and every single experiment utilizing Snapshot ProteomicsTM provides information on >20,000 human proteins without bias to cellular concentration.
-MS/MS requires the use of broad and powerful enzyme inhibitors, and requires that case and control samples be mixed prior to analysis, introducing enzymes from one to substrates from the other.

-The trypsin digestion necessary to MS-based approaches creates variability between samples, and ablates higher order PTM structures.
-Certain PTM-modified amino acid residues have mass/charge ratios (m/z) identical to other unmodified residues, making distinction difficult or impossible.

-10% of the primary amino acids (leucine and isoleucine) show indistinguishable mass/charge ratio and must be inferred from context according to what is already known and present within the database.

*MS-based identification of PTM requires either up-front immunoprecipitation or top- down approaches. The former puts additional experimental variability into a system that started with only 50-70% reproducibility. The latter provides information for only about 1200 of the most abundant proteins.

What are the disadvantages of Snapshot Proteomics compared with MS/MS?

No technique will be the correct approach for all applications. Snapshot proteomics excels in making novel proteomic discovery, but is not the proper tool for targeted needs (detecting pre-identified, specific proteins within complex mixtures). In these instances, we utilize mass spectrometry (MALDI or ESI). Sometimes, none of our products are not the best choice for your project, and we will not hesitate to suggest other companies or technologies to you (e.g., NGS or structural studies).

I have already performed MS/MS analysis on my question of interest. Why should I use Snapshot Proteomics?
Because Snapshot ProteomicsTM is uniquely capable of reporting sample activity and PTM, across a proteomic platform with a depth and breadth of coverage inaccessible to MS-based means. Several clients have found it useful to utilize both technologies, cross-referencing results for added statistical power. We are always happy to perform or assist with this level of analysis.

I don’t have time to chase false leads. Can you guarantee that your hits are real?

We know your time is the most valuable resource you have, and take every precaution to limit false discovery. We can advise you in specific steps that you can take to reduce the likelihood of false discovery. On our end, we use conservative assumptions when analyzing data in order to enrich our results with real leads. Our higher-order data processing reveals pathways, processes, or locations over-represented within the data, adding confidence. Finally, the physiological relevance of Snapshot ProteomicsTM – especially relative to competing methods – is perhaps the best driver of real results.

How are the microchips made? What proteins are present on the microchips?

HuProt arrays contain >20,000 human proteins produced and purified from yeast cells, printed in duplicate on thin-film nitrocellulose-coated glass slides. Randomized deposition of proteins ensures that virtually all epitopes are exposed to the aqueous phase. Purified proteins are printed on the microchips in defined locations, making identification by spatial address straightforward. Various control features enable proper alignment and are also used for statistical normalizations. The array contains broad and fairly consistent coverage (roughly 80%) of human proteins across subcellular location and biological function, including for the membrane proteins. There are splice variants of many proteins, bringing the total number of unique gene ID’s on the array to just under 16,000. The vast majority of proteins are full length, and all bear either GST or His tags. Contact us for a complete list of proteins or questions about specific ones.

Doesn’t immobilization create experimental artifacts?

The vast majority of human proteins don’t work in isolation, rather as parts of dynamic multi-component protein complexes. The points of contact between adjacent proteins within these complexes are naturally extensive, since it is the cumulative effect of many low-strength molecular bonds that maintains the interaction. The native state of a protein within cells is therefore functionally more “immobilized” than that of the proteins on our arrays. Perhaps that is why Western blots, Surface Plasmon Resonance, bead immobilization, etc. are also highly successful and commonly employed approaches. Although randomized deposition of protein to the array ensures that virtually all epitopes are exposed to aqueous phase, it is likely that for most proteins a certain critical epitope IS occluded by the array surface for a sub-population of the members that constitute the feature (or “spot”). However, if 20% of a protein feature is occluded or mis-folded on the case array, 20% of that same protein will be occluded or mis-folded on the control array. This eliminates any artifacts that may arise due the occlusion or mis-folding since the experiments are always differential. Finally, it is worth remembering that no proteins are 100% perfectly folded even within our own cells; estimates from the ubiquitin community suggest that up to 70% of any given protein might be misfolded at any given time, within our own cells.

What is the concentration of proteins on the array?

Each of the >20,000 human proteins are individually purified from yeast prior to being printed on the array. Naturally, some proteins are easier to make than others, and so concentrations of the proteins initially vary. However, because the nitrocellulose surface of the arrays grabs and holds protein at all available and possible locations and because the features themselves are so small (~100 microns diameter), all proteins are present on the array at nearly identical concentration. Mathematically this number is modeled as “infinity”, which precludes our ability to perform kinetic analysis of the arrays proteins, but affords enzymatic amplification of the signals from the enzymes that work on them. It is also worth mentioning that the critical point of comparison in our experiments is not between two proteins on one array, rather the same protein between two (or more) arrays. Thus, if concentrations are slightly variable among the arrayed proteins (due to different levels of initial purity, for example), it is not detrimental to results since protein XYZ on the case array is exactly as concentrated as protein XYZ on the control array.

What do you do with the data you generate for me?

We transfer ownership to you and retain no rights. It is exactly as if you had generated it within your own facility or lab. We hold all client information strictly confidential and will never disclose the names of targets, or even names of clients themselves without permission. We have nondisclosure and confidentiality agreements for use, or will happily execute your own version(s) as desired.

How do I make reference to this work in scientific manuscripts I prepare?

We always disclose the specific (non-confidential) scientific methodologies for each project in manner suitable for publication. You are free to copy directly or paraphrase our detailed methods within the appropriate section of your manuscript. We simply require you to mention that the work was performed by AVMBioMed (Limerick, PA). Alternatively, you may simply state the product that you used (UMAPTM, SMARTTM, or ProActiNTM) with mention of us as the manufacturer (AVMBioMed, Limerick, PA). We do not ask nor expect authorship inclusion; you are never under any obligation or expectation (from us as least) to publish, discuss, or release the data we generated for you.

What is the turnaround time on my job?

This varies by size and scope of the project. For most projects, we will have your data and final report returned to you within four weeks of receiving your materials and deposit. Expedited turnaround can be arranged prior to commencement of studies.

How much do your products cost?

Our products are customized to individual needs, so prices vary by the time and complexity involved with each project. When you contact us, we will schedule a free, no-obligation consultation to discuss your needs and interests. If we are able to assist you, we will provide a detailed and transparent proposal for the project within 2-3 business days. There are never any hidden fees or cost overruns. After agreeing to the terms, a 50% deposit is required to initiate the work, with the balance due 30 days from the presentation of the final report, pending your satisfaction. If we don’t deliver satisfactory results, you never owe the remaining balance.

AVMBioMed, LLC • 321 Jones Blvd. Suite 106, Limerick, PA 19464

www.avmbiomed.com 844.286.2466 • info@avmbiomed.com
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