When discussing proteomics technology much focus is placed on mass spectrometry, and rightly. As you may know, I am a lover of mass spectrometry and I enjoy talking and writing about it . However, it seems to me that not enough is written about mass spec’s proteomics partner of choice, liquid chromatography (LC). The fundamental approach to identification and quantification of proteins in proteomics is not exclusively about mass spectrometry, it is actually about liquid chromatography-mass spectrometry (LC-MS). The essential technology component of proteomics is separation by LC of incredibly rich and complex mixtures from biological samples, such as cell culture, tissue or blood. They are then coupled to identification based on MS/MS spectra (where intact molecules such as peptides or proteins are fragmented), produced by a mass spectrometer, which can subsequently be matched against a database to produce identification. The intensity of signal in the mass spectrometer can be converted into an amount of peptide or protein. Separation of these rich and complex samples is essential as the mass spectrometer measures mass spectra one after the other (in serial not in parallel) and can only measure, at most, ten or twenty mass spectra per second. Thus, with a complex sample that may literally contain hundreds of thousands of peptides or proteins, one must feed the mass spectrometer peptides at a rate of ten or twenty per second. More than that and it will get a stomach ache. Liquid chromatography enables the mass spectrometer to reach, or get close to, this optimal rate of MS/MS speed. This simple proteomics workflow is illustrated in the diagram below.
It’s all about the LC-MS
A great deal of technological development in the last twenty years or so, on both mass spectrometry and liquid chromatography, has enabled today’s capability to characterise to a practical depth the human proteome in less than a day. In other posts I have described mass spectrometry technology and how that has been a great enabler in many applications areas, including proteomics:
Today I will describe a little about chromatography, especially nano-liquid chromatography (nano-LC) and give an example of how this amazing combination of nano-LC and mass spec is practically impacting you and me in today’s world.
‘Nano’ sounds tiny, unimportant and insignificant. It isn’t.
The ‘nano’ refers to something called the flow rate, which is the amount of liquid flowing through the chromatography column (basically a tube packed with material that performs the separation), and is short for nanolitre. A nanolitre is a billionth of a litre and the flow rate through a nano-LC column is typically of the order of 200-300 nanolitres per minute. If you think about how much liquid you would have in a glass of water say, at perhaps 200 ml, then you can see that 200 nanolitres is 1 million times less than that. Why is the flow rate so tiny? That’s a simple one. At extremely low flow rates like this the mass spectrometer becomes more sensitive and able to detect really small amounts of peptides and proteins. So this may be tiny but in a little while and with a small amount of patience (nano-patience?) I hope you will see why nano-LC is small but mighty.
Let’s separate the hydrophobic from the hyrdrophilic
Simply put, with liquid chromatography we can separate molecules based on their physical characteristics, such as their charge or hydrophobicity for example, and it is indeed hydrophobicity that is typically used to separate peptides and proteins in proteomics experiments using a chromatography technique known as reversed-phase chromatography. Peptides and proteins will bind differentially to the reversed-phase material in the column. In other words, one peptide will be bound more or less tightly than another. A bound peptide/protein can be released from the column by passing a liquid or mixture of liquids through the column that can overcome the peptide/protein's binding to the column. At some point the peptide will ‘prefer’ to be dissolved in the liquid than remain attached to the column. By changing the balance of two solvents (for example water and acetonitrile) during the chromatography separation over time, one can create a ‘gradient’ of hydrophobicity (starting with mostly water and increasing the amount of acetonitrile to become increasingly hydrophobic) to separate peptides and proteins so that one peptide might come off or ‘elute’ from the column (become unbound from the column) at a different time point to another. As the peptides elute from the column they are sent to the mass spectrometer for mass analysis and in proteomics, MS/MS, where they are fragmented. This elution time differs according to the hydrophobicity of the peptide or protein.
How has nano-LC changed recently?
So now we have described the fundamental property of nano-LC of the reversed phase type. The changes that have occurred technologically in recent years have enabled longer and longer reversed-phase columns to be used, and used reproducibly and robustly and easily, in proteomics experiments. Chromatography columns for proteomics have gone from 15 to 25 to 50 and, as of last year, even 75 cm in length. Longer columns can separate more and more peptides and proteins from each other. This allows for more and more complex mixtures to be usefully separated in a single analysis, reducing the overall time taken, since it is now possible that no sample fractionation needs to be performed where each fraction would then need a nano-LC-MS/MS analysis. In addition, nano-LC columns have been developed where the column has been fused with the electrospray emitter (the device which transfers the liquid from the column to the mass spectrometer with an electric current applied). When coupled with higher performance mass spectrometers developed in the same time frame of the last several years, this has enabled proteomics scientists to identify tens of thousands to hundreds of thousands of peptides in tens of minutes to hours of a nano-LC-MS/MS experiment. Thus, proteomics, by nano-LC-MS/MS, has come of age, producing powerful results in minutes to hours, and is enabling wonderful science.
I have tried to capture the wonder and, at the risk of sounding teenagery, ‘awesomeness’ of recent proteomics research in earlier pieces (Mass Spec Fourier Transform-ing the Modern World, Precision Medicine…Precisely, and A New Hope for Early Pancreatic Cancer Diagnosis) but frankly, wonderful science is happening every day of the week and there is always something new to describe and discuss. Just recently in the journal Cell Systems, three papers were published that demonstrate that awesomeness. For a wonderfully written summary of these three papers, what has led up to them and, more importantly, their impact, read ‘Proteomics Moves into the Fast Lane.’ For now, I hope the following description of one of those three will whet your appetite for more beautiful proteomics research enabled by nano-LC-MS/MS. ‘System-wide Clinical Proteomics of Breast Cancer Reveals Global Remodeling of Tissue Homeostasis’ is a bit of a mouthful but it is a highly significant piece of work. I shall be a little lazy and quote the final words of the discussion section of the paper, ‘In conclusion, we present a deep proteomic analysis of breast cancer progression and provide a high-quality proteomic database of the results. These data provide functional insights regarding breast cancer development, which are largely distinct from mRNA-based studies, thus reinforcing the importance of the proteomic approach.’ The work presented by Pozniak et al. demonstrates new information compared with transcriptomics research into breast cancer and indeed they identified clinically relevant key proteins and processes that were associated with tumorigenesis and metastatic spread. They did this by identifying and quantifying over 10,000 proteins by nano-LC-MS/MS from 88 cancer samples using an EASY-nLC 1000 chromatography system with separations performed on a 50 cm column coupled to either a Q Exactive or a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer. This powerful data set will now serve as a fundamental resource for future work.
To summarise, it isn’t all about mass spectrometry in proteomics. The mass spec does get all the glory but to make proteomics practical a mass spectrometer has to be ‘fed’ peptides and proteins by a nano-LC system at a rate that it can handle for both identification and quantitation. The nano-LC is key. Today’s combination of mass spectrometry and liquid chromatography is enabling research that is going to help in the near future enable a vision that includes us all living longer and healthier lives. I am by nature an optimist but in research like this and other research that I have described previously I see a lot to be optimistic about. That cancer moonshot might just work out for us all. It will be enabled by technologies like next generation sequencing in genomics but also I believe by nano-LC-MS/MS in proteomics.
To learn more about nano-LC and nano-LC-MS/MS click here.
As ever, if you have the time www.planetorbitrap.com is a great resource for information on orbitrap mass spectrometers.