In Many Cases, The Sample Can Be In A Form Not Suitable For Analysis: Fill & Download for Free

I’ll give a high-level answer suitable for folks outside the field of proteomics.First, iTRAQ is a brand name for a set of isobaric tags sold by Sciex Inc. Other manufacturers of other solutions that work similarly.Both MRM and isobaric tagging are techniques used in bottom-up proteomics to detect differential expression of proteins in complex mixtures. Typical examples of complex mixtures might be whole cell lysates—maybe two different types of cancer cells—or complex fluids such as blood. These studies are attempting to detect “differential expression” by which we mean that they want to know which proteins differ in abundance between the samples. Because they are bottom-up proteomic techniques, the protein samples are going to be enzymatically digested and then subjected to tandem mass spectrometry.To make things a little easier to describe, let’s say that we have two cancer cell lines, and three biological replicates of each. Each biological replicate of each cell line will be processed separately, so the experimenter has six little microcentrifuge tubes—one sample in each. The protein sample in each tube is prepared and then digested down to a very complex mixture of peptides.In the case of isobaric labeling, a different chemical “tag” is going to be reacted with each sample. These tags covalently bond with the N-terminus of the peptides (and with certain side chains). Tags have the property that they will stay put in the mass spectrometer (during ionization), but will break apart during fragmentation. During fragmentation, each tag creates a reporter ion with a characteristic mass. So if the research adds a different tag to each of our six little tubes, mixes all the samples together, then runs tandem MS (MS/MS), the relative abundance of any peptide identified in each of the six samples can be determined from the relative abundance of the six different reported ions.Such isobaric labeling studies are often run in “discovery” mode. This means that the researcher does not know before the study starts which proteins they will find. As peptides elute of a chromatographic column, they are automatically subjected to MS/MS and then informatics tools are used to infer which proteins were present based of the peptides discovered. Different tools are then used to infer the relative abundance of each protein based of the relative abundance of the six reporter ions of each peptide associated with each protein (with a bunch of statistical details that need to be attended to.) Typically a study of this type might discover several thousand proteins, but there is no guarantee that any specific protein will be discovered.In an MRM study, a fixed set of proteins are selected and an MS “method” is developed for each protein. In these methods, peptides are identified that have a rare intact mass (and possibly LC retention time). Next, from those peptides unique fragment ion masses are determines such that if the first mass is detected and fragmented, and the second mass is then observed, in can only mean that the protein of interest was in the sample. The method will typically have several dozen different proteins that it will detect. For our cancer example, the researcher might be interested in KRAS and several other cancer-related proteins. Once the MRM method is created, samples can be quickly screened for the proteins of interest—and the intact intensity can be used to determine either the relative or absolute abundance of the protein (depending on things outside the scope of this answer.)Typically MRM studies take a lot of work to develop the method, but once done, they can be quickly run on many samples—far more than the six in our simple example. But they only report on the proteins targeted by the method.

Please direct me to the place where “perfect science” exists. Other than on a chalkboard, science is never perfect. I have worked in several labs and the results of any analysis always have an error bar. There is never a perfectly exact answer to any lab analysis.Carbon dating, and radiometric dating in general, is very reliable and accurate. It is not perfect. There are several limitations to carbon dating.Carbon dating only works on items that were once alive. The process assumes the object being dating was in equilibrium with atmospheric carbon when it died. So, only living organisms.Carbon dating only works on items that gained most of its carbon from the atmosphere. The process assumes the object being dating was in equilibrium with atmospheric carbon when it died. A few organisms like clams may take most of their carbon from geologic sources, limestone, and are not suitable for carbon dating.Carbon dating only works on items that are less than about 60,000 years old. Since the half life of carbon-14 is 5730 years it is mostly gone after that.Carbon-14 levels in the atmosphere vary slightly over time. A correction curve is generated to adjust for times high or low solar activity.On particularly old samples, contamination with current carbon-14 must be avoided.The atmospheric carbon-14 levels has spiked since 1945 due to nuclear testing. That makes it useless for organisms that died since then.You need a very sensitive mass spectrometer.You must carefully guard to minimize contamination. This is particularly true of very old samples where just a few atoms of carbon-14 from today’s environment can really throw off the results.The technicians must have calibrated their equipment properly.The technicians must do their work properly.

Yes, we get meteorites from Mars, but no samples returned by our spacecraft.Natural sample returnYou can imagine, it must be a pretty huge impact on Mars to send rocks all the way to Earth. With a small impact all the material stays on Mars, and for a big impact too, most of the material returns to Mars. But if the impactor is more than one kilometer across, creating a crater more than ten kilometers across, some surface rocks can reach escape velocity. They end up in a similar orbit to Mars, but after some fly-bys of the planet, a few of them can get diverted to impact on Earth.So, though most are never recovered there are at least several meteorites land on Earth every year from Mars. But, they all come from a small number of impacts, as you would expect,You can tell that by measuring the time spent exposed to cosmic radiation, which confirms the conclusions of the model,Measured ages so far are (time since the meteorite was ejected from Mars) are 20, 15, 11, 4.5, 3, 1.3 and 0.7 million years.(see Martian Meteorites - Cosmic Ray Exposure Ages and Figure 9 of scholarly paper)Life on Earth could have originated on MarsThere was a lot more exchange of material in the early solar system, and one theory of the origin of life is that it could have started on Mars and been brought to Earth by meteorites. It's possible because Mars, further from the sun, cooled down sooner, and was habitable at an earlier stage than Earth. Also had no Moon forming giant impact to disturb it early on.It had oceans, for a few hundred million years, then massive flooding for another few hundred million years. Perhaps that was enough time for life to arise, as it seems to have got off to a very early start on Earth. Some rocks could get from Mars to Earth within a century, and some micro-organisms are hardy enough to survive that, modern ones anyway.Our meteorite samples are limitedWe do have a reasonable spread of ages, the radiometric ages(age since formation) range from 4.5 billion years for Allan Hills 84001 to a few hundred million years in the case of the younger meteorites.However, most are igneous rocks. Our rovers and satellites have found many other rock types on Mars and these haven't been found yet on Earth as a Martian meteorite.Spacecraft sample return ideas:So there is a lot of interest in sample return from Mars to Earth, to take a closer look at whats there. But there are environmental issues. The problem is, the most interesting samples would be ones with life in them, past or present. But if there is present day life in the sample, maybe it could have some harmful impact on Earth?Also some exobiologists say we don't know enough yet to return biologically interesting samples to Earth.The idea has been suggested many times dating back to the 1970s, this is from a 1978 study:The problem is that the sample has to be carefully contained. First, you have to keep Earth micro-organisms out of it. A single Earth microbe or virus, even a single amino acid from Earth would be enough to invalidate modern sensitive analysis of the sample. If you find amino acids in the sample for instance, you have to be sure they are from Mars and not contamination from Earth?But the main issue is protection of the Earth. The thing is, we have no idea if there is life on Mars or not, and what the nature of it is if there is.Actually since Phoenix there's been new interest in the possibility of life on Mars. These would be micro-organisms living on the edge, in a desert like landscape, like the dry valleys in Antarctica. They could live on drops of water melting briefly around a grain of sand in the snow. Or might be like arctic lichens able to live just off the humidity in the air which is close to 100% on Mars when the sparse frosts form in the morning and evening. Or below the surface of rocks. Or they could live in millimeters thin layers of water that probably form when certain salts deliquesce. See my Might there be Microbes on the Surface of Mars?If there is life in the sample, then until we can study it, there is no way to know if it is safe to return it, for sure. The experts are generally agreed that it is almost certainly going to be of no harm at all to Earth. But the worst case, thought to be very low probability scenario is severe in the extreme, leaving quite a dilemma as to what to do.Carl Sagan wrote in his book Cosmic Connection:…Precisely because Mars is an environment of great potential biological interest, it is possible that on Mars there are pathogens, organisms which, if transported to the terrestrial environment, might do enormous biological damage - a Martian plague, the twist in the plot of H. G. Wells' War of the Worlds, but in reverse. This is an extremely grave point. On the one hand, we can argue that Martian organisms cannot cause any serious problems to terrestrial organisms, because there has been no biological contact for 4.5 billion years between Martian and terrestrial organisms. On the other hand, we can argue equally well that terrestrial organisms have evolved no defenses against potential Martian pathogens, precisely because there has been no such contact for 4.5 billion years. The chance of such an infection may be very small, but the hazards, if it occurs, are certainly very high.All the official studies since then have agreed with Carl Sagan's conclusion, that it is necessary to take precautions against that possibility.I've found it hard to talk about this with some folk, because there is a tendency to assume it is about some weird Andromeda Strain type alien,But as you see, Carl Sagan's concern was about something much more prosaic. A disease like Legionaires disease perhaps, but one we aren't adapted to. Or, a virus that attacks micro-organisms (because of possibly shared ancestry way back). Or a micro-organism that out competes our native life - a bit like the way that rabbits out compete marsupials in Australia. Or maybe it damages our crops, or puts natural cycles and ecosystems out of balance. Or it's spores irritate our lungs, or some other type of an allergen.Does the natural sample return make it safe to return a sample to Earth?The idea of life transferred to Earth on the Martian meteorites is new since Carl Sagan's day. Perhaps life might occasionally transfer from Mars to Earth via meteorite. If so - does that mean that any sample we return from Mars is harmless?Robert Zubrin thought so as have a few others since then. But his is a minority view on this topic. Exobiologists particularly don't agree with his views on it.The thing is - any transfers of life are going to be rare.Impacts on Mars are only every few million years or soLife most likely to make the transition on meteorites that reach Earth soon after the impactImpacts mostly on igneous rockNo direct evidence of life on meteorites from MarsSo, if there were any extinction events or obvious changes in our environment caused by transfer of life from Mars, they would be rare, so hard to spot. There have been many extinction events in the past not fully understood.The National Research Council looked into this and concluded that though there have been no large scale effects on Earth likely to be caused by life on meteorites in recent times, the possibility can't be ruled out further in the past.They also noted that there are many forms of life that wouldn't be able to make the transition on a meteorite, but able to get here in the carefully preserved environment of a return capsule.Also the meteorite has to hit a habitat on Mars containing life - if this is a micro-habitat consisting of a thin patch of salty brine in the sub surface soil, would this survive a meteorite impact and would the life be sent unchanged all the way to Earth?In a sample return canister, the samples would be carefully selected, and carefully preserved for the journey.Present day suggested precautionsThis is a more modern version of the official precautions, we have moved forward a long way since Antares.Recent ideas suggest return to Earth itself, and e.g. this particular version has robotic handlers:It is an expensive to build building - and the first ever facility of it's type so will probably have "teething difficulties".It requires you to combine the clean room technologies, which use positive air pressure to keep micro-organisms out, with the biohazard technologies that use negative air pressure to keep them in. Most proposals end up with a triple walled structure to handle both of those at once.The official studies did estimates of the time needed to get it designed, built, and up and ready with staff familiar and able to operate it without incident, they came up with figures of over a decade before it is ready to receive a sample return.Needs to contain unknown biohazards of unknown sizeThen you have to contain an unknown biohazard, so you don't know how small they are. This is different from the usual situation, to contain known biohazards.To be as safe as possible you design it to contain micro-organisms half the size of the smallest known ultramicrobacteria. The size of confirmed ultramicrobacteria has gone down to 0.2 microns and may go down further. So their recommendation was that any release of a particle larger than 0.05 microns is unacceptable.Then there is the possibility of the even smaller Gene Transfer agents to think about. These are as small as 0.01 microns across, and could transfer DNA between micro-organisms if the life on Mars uses the same DNA mechanisms as Earth life does. In one experiment, then GTAs left overnight in a sample of sea water transferred DNA to half the micro-organisms in the sample. So - this is quite hard to guard against.Of course you can contain it if that is all you want to do. The problem is, how can you do experiments on the sample, cut sections off it, move bits around in the facility, and at the same time make it impossible for sub micron particles to ever encounter the Earth environment, or any Earth DNA or amino acids etc. to ever encounter the sample?Then, if it is based on a completely different biochemistry the whole thing is basically guesses about what size it is, and what effects it could have.Return sterile samples onlyOne solution of course is to simply sterilize the samples before you return them to Earth. Then there is nothing to worry about. The problem is of course, that that also destroys most of the interest of the samples.Another idea is to return samples of past life only, not go anywhere near regions on Mars that might have present day life. But there you have the issue that first, some micro-organisms may be able to remain viable for millennia on Mars, as they do on Earth, and the global dust storms spread material from all over Mars .So the dust could have viable spores and other dormant life hidden in cracks, protected from UV by the iron oxides in the dust.Also there are some ideas for life able to survive even in the driest regions of Mars, around deliquescing salts, in the "advancing dunes bioreactor" model.Need for public debate and legislation changes for potential of environmental disruptionThis possibility also means that in the low probability worst case scenario, the nations affected by the sample return could include any of the nations on the Earth and not just the nation responsible for launching the mission.So apart from any legal requirements, morally, any nation that does a MSR should involve other nations in the debate. It's not right for one nation to unilaterally take this risk however small it might be.This also makes it a major legislative tangle. Things were much simpler legally in the Apollo days.NASA released its quarantine proposals for Apollo 11 on the same day as the launch leaving no time for public appraisal of them. That would not be tolerated today. The Apollo era quarantine rules were rescinded so can't be used as is.Also many of the modern environmental laws didn't exist in those days.Today there would be many domestic US hurdles (if it was a NASA mission), which would take many years to work through. Also,it would be subject to international agreements, and domestic policies of other nations.So, there would need to be a big multi-nation process of new legislation before a MSR could be approved.Margaret Race of the SETI institution went into this in detail, I summarize her findings here:Legal Issues and Need for International Public DebateWhat if the samples are biologically uninteresting?Some exobiologists put forward this argument strongly.The thing is - that what we are most interested in is evidence of life on Mars. But the evidence so far suggests that life on Mars, if it exists,is going to be hard to find.Present day life on Mars might exist in micro-habitats, just a few mms in thickness, and maybe only exist for part of the day or year. Over the entire surface of Mars maybe some of these habitats have life and some don't. For instance it might depend on a delicate balance of salts, and then the life has to find the habitat to colonize it.They may also have low quantities of life, just a few micro-organisms metabolizing really slowly and occasionally reproducing, perhaps even only reproducing every thousand years or so as happens for some extreme environments on Earth.That's because of the low temperatures and low levels of essential materials. It's probably similar to present day extreme desert and Antarctic dry valleys habitats on Earth, but may have even lower populations.This could be beyond detection capabilities of anything except the most sensitive instruments, beyond anything sent to Mars to date. So we wouldn't know if there is life until the sample is returned.Or life on Mars could be deep underground, hard to reach.Same issue applies to past lifeAncient life, to be well preserved long term, needs special conditions. Salt or clay deposits are best, and at least several meters below the surface (because it gets degraded over geological timescales by cosmic radiation). These samples would be hard to reach, and quite possibly only some of the apparently suitable samples actually include any life signatures in them. You would be lucky to find evidence of life in your first deep drill sample on Mars.Results likely to be inconclusiveSo whether there is present day life, or was life in the past, or no life, whatever the situation, seems unlikely a MSR at this stage would do all that much to help settle the question, unless you get really lucky and happen to return a sample that has life in it.A Mars sample return could easily end up returning biologically uninteresting samples, at great cost. Since the search for life is the main reason given for attempting a MSR it seems there is at least quite probable that it ends up as an expensive mission that does little to help increase our understanding of its main objective.Better in situ instrumentsSeems a better approach to investigate it on the surface first with more sensitive instruments. There are many proposals for new life detection instruments of very high sensitivity small enough and light enough to fly to Mars. E.g. the "astrobionibler" sensitive to presence of a single aminio acid in a gram of sample, also scanning electron microscopes, DNA sequencers and the like, all under development.Alternative, teleroboticsThis is what I favour myself at present.Rovers on the Mars surface operated by telepresence by humans in orbit.Time delay issues from EarthThe problem for exploration from Earth is the time delay of many minutes. No-one can drive the rovers in real time so they have to proceed extremely slowly. They have feeble motors that can only drive slowly but that is mainly because there is no incentive for them to be faster.Human mission to Mars orbit for telepresence operationFor the same cost as a sample return to Earth, bearing in mind all the expenses of the entire mission including receiving facility on Earth etc, you could probably send a human mission to Mars capture orbit. This is an elongated orbit, not the low orbit which is expensive to reach in terms of fuel. It swoops in to Mars, passes close by, and then swoops out again. Turns out to be a good orbit for telepresence operation In terms of delta v, it is as easy as a Moon landing.At the same time, you land lots of rovers on Mars, operated by the humans in orbit around the planet via telepresence..Have sensitive life detection instruments on those rovers. In a day the humans in orbit with their telerobots could do as much as we now do in years. In a mission lasting a year or so we would advance our understanding of Mars immeasurably.At that point we could start to think about whether a Mars sample return is a good idea. We would know a lot about Mars by then, know which samples are biologically interesting, know exactly why we want to return it, and have a good idea of whether it is likely to be biohazardous and what is the best way to contain any hazards.I would do biohazard testing in orbit around Mars too at that point, e.g. in greenhouse type hab, small satellite orbiting separately from the human mission to test effect on environment of the samples in a preliminary way (especially if any biosignatures are found) - why not take as many precautions as you can.That's my opinion on it, for discussion.Ideas of telerobotic explorationThis idea has been suggested many times, including HERRO, and Robert Zubrin with his Athena double flyby, also a Russian proposal and one by Lockhead Martin.Maybe it's time has come though. Last year there was a major telerobotics conference supported by NASA that went into this and they concluded that if there is a human mission to Mars orbit, it would be a case of missing a major opportunity if they don';t explore it via telerobotics.I go a bit further and say we should keep to telerobotics only in the near future, no human landing until we know what the surface is like, what is the hurry?Also, I simply wouldn't bother about a sample return until we have done a fair bit of telerobotics.Bearing in mind the arguments of the exobiologists, there doesn't seem to be much point to do it now - not enough to justify the huge cost of the missionChances are that it is totally safe, but why take any risk at all?As Carl Sagan said in Cosmos“ If we wish on Earth to examine samples of Martian soil for microbes, we must, of course, not sterilize the samples beforehand. The point of the expedition is to bring them back alive. But what then? Might Martian microorganisms returned to Earth pose a public health hazard? The Martians of H. G. Wells and Orson Welles, preoccupied with the suppression of Bournemouth and Jersey City, never noticed until too late that their immunological defenses were unavailing against the microbes of Earth. Is the converse possible? This is a serious and difficult issue. There may be no micromartians. If they exist, perhaps we can eat a kilogram of them with no ill effects. But we are not sure, and the stakes are high. If we wish to return unsterilized Martian samples to Earth, we must have a containment procedure that is stupefyingly reliable. There are nations that develop and stockpile bacteriological weapons. They seem to have an occasional accident, but they have not yet, so far as I know, produced global pandemics. Perhaps Martian samples can be safely returned to Earth. But I would want to be very sure before considering a returned-sample mission.”So, maybe it can be done safely with modern technology with great expense and care. But my view is, why such a rush? Let's do what we can "in situ" first.Find out moreCan Human Explorers Keep Mars Clean, For Science?How Valuable is Pristine Mars for HumanityMight there be Microbes on the Surface of Mars?Need For Caution For An Early Mars Sample Return - Opinion PieceMars Sample Receiving Facility and sample containmentLegal Issues and Need for International Public Debate