Mittwoch, 8. August 2018

In vivo TMT labelling

Tandem Mass Tag (TMT) is a common technique for quantification of peptides at the MS2 level.
The TMT is based on the reaction of the Primary amine at the N-terminus and the lysine with the NHS-ester group of the isotopically labeled tag. Once successfully tagged the peptide mixture is subject to MS analysis were the TMT-peptide displays a label-specific, low mass reporter ion during MS2.

The NHS-based TMT strategy requires all peptides to be freely accessible within a lysate to obtain efficient tagging. TMT can be apply for intact proteins as well, but applying it to intact cell in vivo was new to me.
The authors investigated the labelling efficienicy among different cancer cell lines and stated that in vivo TMT labeling requires an additional enrichment step to achive decent labeling efficiencies which are still lower compared to tagging on the peptide level (roughly 50% of identified peptides were tagged with invivo TMT after enrichment). The enrichment was done using an anti-TMT antibody to pull down all labeled peptides.

Compared to TMT labelling on the peptide level which appeared to be 100% of all identified peptides in vivo labelling with subsequent
Tandem Mass Tag (TMT) is a common technique for quantification of peptides at the MS2 level.
The TMT is based on the reaction of the Primary amine at the N-terminus and the lysine with the NHS-ester group of the isotopically labeled tag. Once successfully tagged the peptide mixture is subject to MS analysis were the TMT-peptide displays a label-specific, low mass reporter ion during MS2.



The NHS-based TMT strategy requires all peptides to be freely accessible within a lysate to obtain efficient tagging. TMT can be apply for intact proteins as well, but applying it to intact cell in vivo was new to me.
The authors investigated the labelling efficienicy among different cancer cell lines and stated that in vivo TMT labeling requires an additional enrichment step to achive decent labeling efficiencies which are still lower compared to tagging on the peptide level (roughly 50% of identified peptides were tagged with invivo TMT after enrichment). The enrichment was done using an anti-TMT antibody to pull down all labeled peptides.
Compared to TMT labelling on the peptide level which appeared to be 100% of all identified peptides in vivo labelling with subsequent immunoprecipation showed rather minor efficiency. However reproducibility was definitely given. Surprisely, the in vivo labelling strategy had no specificity to the location of the proteins. Specifically, a bias towards surface protein has not been revealed.




 showed rather minor efficiency. However reproducibility was definitely given. Surprisely, the in vivo labelling strategy had no specificity to the location of the proteins. Specifically, a bias towards surface protein has not been revealed.



Freitag, 3. August 2018

New features in skyline - LC-IMS-CID-MS and contaminations library


There is a lot going on in the skyline world lately. Originally, open source software skyline from the MacCoss Lab which started out as analysis tool for label-free quantification and MRM analysis of MS data.
Over years a lot of features have been added because more comprehensive analysis was requested by the users or new instruments using new scan modes have been introduced to the market. That's why recently ion mobility functionality has been added, including the entire LC-IMS-CID-MS workflow.


However, yet another skyline feature caught my attention in the current JASMS. A contamination library has been integrated into skyline. It contains over 684 common MS contaminations, which can be used as an transition list for MS1 filtering and is also provides a approach to determine unknown contaminations via a mass-to-formula tool. The mentioned transition list can be downloaded in the public repository panorama.


CID and CIU

CID stands for collision-induced-dissociation a common fragmentation technique to create tandem MS spectra (MS2). In CID precursor ions are accelerated and subsequently injected into a ion guide filled with inert neutral gas molecules, typically N2, where the analyte of interest undergoes single or multiple collisions. The charge of the analyte, the accelerating (collisional) voltage and the gas pressure can determine the extent of collisional Impact.

Ions are vibrational excited by the collision(s) with a time frame of a few femtoseconds. Depending on the chemical bonds present the ions can break apart into charged, radical or neutral fragments. In proteomics weaker bonds, such a post-translational modification, tend to get lost during CID.
In beam type instruments a special version of CID, In Source CID, can facilitate MS3 fragmentation for deeper structural elucidation. Herein, ion guides on the front end of the MS serve as a 2nd collision device for fragmentation of all ion species injected. Out of this very complex MS2 spectrum precursors can be selected for an MS3 in the actual collision cell downstream.  

When it comes to analysis of intact proteins and protein complexes having high molecular weights, In-Source CID at elevated pressure, can be applied but in most cases it directly leads to dissociation. Secondary or high order protein structures cannot be detected easily.
Besides the dissociative nature of CID for large biomolecules there is a much elegant appoarch that utilizes collisional activation to study structures intactly. It is called CIU, which stands for collisional induced unfolding, and can be conducted with an ion mobility detector coupled to an MS.
During a CIU experiment intact biomolecules, such as antibodies, are ionzied and undergo a stepwise increase of collision heating which induces a gradual change in conformation (unfolding).
For every stepped potential an ion mobility scan, plus the nested MS scans, are recorded so that one can follow the structural changes. These multidimensional datasets (see image) help to distinguish isoforms, determine number for disulfide bonds and degree of modifications or monitor ligand binding.

Source: https://www.sciencedirect.com/science/article/pii/S1367593117301266?via%3Dihub#fig0010

Dienstag, 31. Juli 2018

CharmeRT - Chimeric spectra identification utilizing retention time prediction - Up to 50% more peptide IDs


Co-elution of peptides is a major issue in bottom-up proteomics experiments, since it can led to co-isolation and co-fragmentation of peptide ions resulting in a chimeric MS2 spectra.  A MS2 spectrum containing more than a one peptide spectrum match (PSM) is considered to be chimeric MS2 spectra.
By the way chimera is taken as an analogy from greek mythology, where it stands for a hybrid creature from two or more animals. An example would be pegasus, the winged horse, so basically a mixture of a horse and a bird. 



But now back to the problem having multiple peptides selected as MS2 precursors… According to the study a HeLa digest separated by a 3h gradient displayed over 50% chimeric MS2 spectra, 20% of these spectra showed even more than two PSMs in a single MS2.
Sounds awesome to me and really is a reason to further investigate - how the authors have done this.
Well, as it turns out they identified the chimeric spectra by performing a 2nd search, in which previously assigned PSMs have been removed from the raw data. These multiple PSMs were then validated utilizing retention time prediction model as part of Elutator software. This prediction model is based on the hydrophobicity index of the (modified) peptides. The index determines how much energy is needed to transfer molecules (or a peptide chain) from a nonpolar solvent to water.
If the polypeptide requires energy to do this it is hydrophobic. If it does not it is considered to be hydrophilic. In reverse phase liquid chromatography such an index can provide a measure of approximate elution order and time.


The special feature of Elutator is that it not only takes the hydrophobicity of the individual amino acids into account but also it considers the effect of neighbouring amino acids during its calculations. This lets Elutator predict retention times much more precise compared to other RT prediction tools.


CharmeRT using Elutator outperformed conventional proteomics workflows such as mascot and percolator by up 90% in terms of assigned PSMs and up to 65% for validated peptide IDs. It is claimed that most of these newly identified peptides are low abundant peptides.  Therefore the authors tried to investigate this hypothesis with RNA data of the identified peptides. And indeed, they showed an extension in sensitivity towards smaller peptide abundancies without increasing instrument time, well just post processing time, though.
Charme RT can also be beneficial for DIA approaches, since it works much better for wider quadrupole isolation windows.


Mittwoch, 25. Juli 2018

Temperature Gradients in on-chip LC provide an solvent saving alternative

Lately, I have been reading a lot about lab-on-chip technologies and I am beginning to admire the advantages of this technology. 

However....Temperature can be parameter targeted by method optimization to speed up liquid chromatography. Generally, it is known that temperature increase leads to an increase of brownian motion and causes faster separation (lower retention times (k) but also peak broadening and a change in selectivity due to differences in mass transfer.
Other positive side effects are lower backpressure which enables the usage of longer columns or smaller particle to increase efficiency and lower solvent consumption.


This dependency can be explained by the Horvath Rule, named after the hungarian Csaba Horvath who is known for a lot inventions in the field of LC, for instance pellicular particles. The rule points out that an temperature increase of 4-5 degree celisus has similar effects as increasing the organic modifier by 1%.

When it comes to temperature gradients in conventional LC and peak broading one has to differentiate between an radial and axial gradient. The radial gradient (in to out) causes intense peak broadening, whereas the axial gradient (front to back) leads to peak shapening and decrease retention times. In tradional LC pre-column heater and post-column cooler can prevent these effects related to the so called thermal mismatching.

If you are performing LC on chip device the interface with the heater is much larger compared to an conventional LC column and therefore one is going to have a much faster thermal response time. Solvent pre-heating is obsolate. Heat dissipation is reduced since there is no air contact. 


Due to dependency Horvath has described one does not need a solvent gradient to increase peak capacity for on chip LC temperature gradients one can be used instead. This saves ressources and reduces the ecological impact of analyical labs. 

https://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b00142

Portable mass spectrometers utilizing paperspray ionization and LT plasma desorption

Miniaturation or downscaling of mass spectrometer is a growing field, especially in the space and military sector. Operators should be able to carry the devices around to do on-site analysis of various substances, such as toxins, pesticides or explosives. Within this project students from University Purdue took their portable gadgets to the grocery stores to proof the real life application.




Such projects are awesome - I wonder how customers reacted?

In this case two ionization methods were performed - plasma ionization and paperspray ionization. During paperspray ionization a sample is wiped with an solvent-wetted tissue, which is later cut into a small triangle in order to produce a electrospray at the tip when HV is applied to it.



The second method, called low temperature plasma ionization (LTP). The LTP probe produces long lasting meta-ions (plasma) from reagent gas (He,N2) via a low frequency AC voltage induced discharge afterglow. The afterglow is usually a sign of the presence of excited particle species. These particle ionize molecules on the sample surface, which are then desorbed by the MS. LTP ionization causes less radical formation and analyte fragmentation compared to APCI. In contrast to DART,  LTP plasma is not heated up to facilitate sample desorption.

Dienstag, 24. Juli 2018

Are we all becoming mass spec extremists?

Recently, I read an article from 2013. Within this article Graham Cooks, a pioneer and well-known mass spectromety researcher from Purdue (see image), said that he takes the "extremist view" that such separations [LC, GC or CZE were meant here] shouldn't be necessary. Instead of pre-separating mixtures, they can be ionized and the ions separated in a mass analyzer. Fragmentation and multiple stages of mass analysis can then be used to characterize the ions.

Bildergebnis für graham cooks

Now, 5 years later we can admit that instruments have been developing more and more in this direction. Developers try to optimize duty cycles, perform on-the-fly calculations to prevent oversampling and increase peak capacity at the same time.
A current example for this is recent trend coupling liquid chromatography, ion mobility and mass spectrometry.

During ion mobility ions are separated based on their spatial extent. Many techniques have been developed to determine (reduced) ion mobility, K0, which can be converted by Mason-Schamp Equation to  collisional cross section values (omega in Angstroem squared).

The easiest technique is the drift tube technique, in which a mixture of ions is subjected in the tube having a static pressure of 1bar and less. The separation is based on their mobility which is caused by friction originated from surrounding gas molecules. Herein, compact ions displaying high mobility and elute first, whereas large ions elute later. Usually it takes about 10ms to scan the entire ion mobility range.

The millisecond scan time fits perfectly into the time dimension of the elution time of a chromatographic peak, which usually takes seconds and a TOF scan, which takes microseconds. The additional information of ion mobility (in addition to retention time, isotopic pattern, m/z and intensity) provides a tool to analytical scientists to unambigueously identify isobaric and stereomeric compounds, which otherwise would overlap in MS-only analysis.


Prospectively, I am trying to work on posts covering various applications and the newest technological developments in this new branch of hyphenated mass spectrometry. I am looking forward to it!

What is Admiral Ackbars favourite Mass Spec?



This is some trash talk! I really like the Star Wars Saga. This scene is from episode VI - return of the Jedi and even this hollywood masterpiece can be related to mass spectrometry.

https://www.youtube.com/watch?v=4F4qzPbcFiA

Medronic acid improves detection limits of phosphopeptides

Phosphopeptides, organic acids or nucleotides are acidic and can gain a negative charge in the liquid phase. When it comes to LCMS analysis this charge introduces a second dimension of selectivity and causes tailing in HILIC chromatography, especially in presence of positively charged metal ions (low purity silica).


Formly chelating properties of EDTA have been used to remove most metal ions but this additive caused ion supression in downstream ESI ionization. To overcome this incompartibility with ESI MS analysis the use of medronic acid was investigated. As it turns out medronic acid has a much higher affinity for metal ions than phosphopeptides and phosphorylated compounds and displaying much less ion supression compared to EDTA in ESI MS.

https://pubs.acs.org/doi/10.1021/acs.analchem.8b02100

Sonntag, 22. Juli 2018

MALDI 2 enhances sensitivity by two orders of magnitude

MALDI 2 is a further development of MALDI, in which laser energy is applied to a solid analye-matrix sample. The analyte-matrix cluster absorbs the laser energy and gets ionized, ablated and protonated within the gas phase.


Two theories have been proposed for these processes. The lucky survivor theory in which the analyte is already charged before it is released into the gas phase. Here counter ion reactions with the oppositely charged matrix ion can take place and form a neutral cluster which can not be detected anymore. Only the analyte ions which do not react with the matrix will make it to the detector.

The 2nd theory - assumes that the analyte is evapourated into the gas phase as neutral and gets gas phase protonated by the matrix.

The two theories have in common that there are a certain number of neutral matrix or matrix-analyte cluster present in the gas phase. These neutrals are lost within the MS but still have the potential to be ionized and therefore limit the ion yield.
MALDI 2 tries to target exactly these neutrals, which require more energy to ionize or to re-ionize, by inducing a second MALDI ionization.

To minimize spatial expansion of the ion plume source parameter, such as pressure, laser wavelength, beam focus and the timing are really important to make this technique work. Since spatial expansion is still present and therefore different starting conditions for the MALDI ions orthogonal TOF is highly benefical for mass analysis.

MALDI 2 increases sensitivity for lipids by two orders of magnitude.
http://science.sciencemag.org/content/early/2015/03/04/science.aaa1051.full