Determinationof Ubiquitin`s Molecular Mass Using Electrospray Mass SpectrometryTechnique

Electrospraymass spectrometry is a molecular biochemistry technique that has beenused to determine the molecular mass of different biomolecules. Theseinclude proteins and peptides among other macromolecules present innature. The determination of the molecular weight of variousmacromolecules is necessary as it helps in experimental analysis andstudy of organism’s structure and functions. The other methods thathave been used to quantify biomolecules include infraredspectrometry, gas chromatography, and mass spectrometry among others.Electrospray mass spectrometry has been defined as a soft ionizationmechanism that analyses bimolecular weight of substances based ontheir ionization potential. The method can only analyses biomoleculesof larger molecular mass as it has no ability to fragment moleculesto small charged particles. The technique involves ionization ofmacromolecules into small droplets which excite protons. Theprotonated and dissolved molecular ions are directed via the massanalyzer as well as the detector, which determine the sample mass(Murphy).

Theanalysis is normally conducted using a mass spectrometer, which hasan ionization source, the mass analyzer, as we as a detector. Thistechnique utilizes the soft ionization technique, unlike the hardionization technique that involves electron impact ionization andfragmentation in Gas chromatography-mass spectrometry method. It isbelieved that electrostatic repulsion between charged particlesinside the capillary of the spectrometer induces the particles toenter a gas phase. These molecules present in solution are normallyionized and un-fragmented resulting to the difference in mass chargedhence different mass spectrum. The mass analyzer uses strengths ofapplied fields to determine the mass of ions that passes through it.The detector counts ions at high energy thereby determining themolecular mass of a sample.

Proteinshave always been analyzed using these techniques because of theirstructure composed of amino acids. The hydrophilic and hydrophobicnature of amino acids provides a charged ion states of thebiomolecules. Mass Spectrometry depends majorly on the chargedproperty of amino acids and the basic side chain that defines theprotein charges. Since pH of different solvents affects the aminoacid charges, it is also important as it affects mass to chargeration of proteins during quantification (Lab 7).

Quantitativeanalysis of the results relies on mass to charge ratio (m/z) ofdiverse peaks present in the spectrum. A spectrum has always beenobtained with a mass to charge ratio plotted on the x-axis inaddition to the % (percentage) relative intensity of every singlepeak plotted on the y-axis. The unknown mass, Mr, of the biomoleculemay be determined based on spectral data as p = mz that is




Fromthe equation, p1 and p2 are regarded as the contiguous peaks. Studieshave indicated that the main advantage of this technique over othersinclude its ability to handle large masses of samples, and the softionization method that allows it to effectively analyze biomoleculesdefined by non-covalent interactions. The method can also indicate asample structure accurately. The instrument is also highly sensitiveand thus the qualitative and quantitative measurements can accuratelybe conducted for different macromolecules.

Themethod, however, has some disadvantages that include its inability toanalyze a mixture of molecules accurately. The instrument has atendency of frequent contamination and is not easily cleanable.

Themain aim of this experiment is to determine the molecular mass ofubiquitin using electrospray mass spectrometry technique. This willbe conducted by running ubiquitin through the mass spectrometer toobtain its mass spectra under different solvent conditions. The otheraim of the experiment will be to determine the effects of pH onprotein stability. This will be conducted through the analysis of themass spectrum of ubiquitin at different pH values. The experiment isalso aimed at determining the effects of the organic solvents onubiquitin stability.


About10-3 M ubiquitin stock solution was provided in water. A 1ml dilutionof 10 to 6M ubiquitin was prepared through dilution of the stockubiquitin in every single of the following solutions. The firstsolution had 20% acetonitrile and 80% Acetic Acid (pH=2.75) as wellas water. The second one had 5% acetonitrile and 95% acetic acid(pH=2.75) with water and the third solution had 20% methanol and 80%acetic acid (pH=2.75) plus water. The last solution had 40% methanoland 60% acetic acid (pH=2.75) with water.

Thesesolutions were then run on Varian 325-MS mass spectrometer. Thesamples were run on the spectrometer using a `115 50-50 posneg 4 min`method. Chromatogram as well as mass spectra for every single &quotpeak&quotin the chromatogram was obtained for the determination of themolecular mass of ubiquitin and other analysis. The arrangement ofpeaks in the mass spectrum of the pH=2.75 sample was used to computethe molecular mass of ubiquitin.


Figure1: Chromatogram and spectrum of ubiquitin run with 40% methanol at pH2.75

Basepeak = 98.8 (1.061e +8=100%.

Therelative intensity attained was less than 5% for 40% methanol runwith ubiquitin at pH 2.75. The figure indicates that electronsdecreased as the mass to charge ratio decreased. The relativeintensity, however, was decreasing with an increase in mass to chargeratio of the particles for the peaks formed.

Figure2: Chromatogram and spectrum for ubiquitin run with 20% methanol atpH 2.75

Basepeak = 98.8 (1.534e +8=100%.

Therelative intensity attained was 10% for this run that involvedubiquitin mixed with 20% methanol organic solvent. The figureindicates that the higher the mass to charge ratio, the lower therelative intensity attained and higher protons generated.

Figure3:Chromatogramand spectrum for ubiquitin run with 5% acetonitrile at pH 2.75

Basepeak = 98.8 (1.271e +8=100%.

Therelative intensity attained was 12.5% for ubiquitin run with 5%acetonitrile organic solvent at pH 2.75.

Figure4:Chromatogramand spectrum of ubiquitin run with 20% acetonitrile solvent

Basepeak = 98.8 (8.472e +7=100%

Therelative intensity achieved was less than 10% for the run thatinvolved ubiquitin mixed with 20% acetonitrile at pH 2.75. Thisindicates that the concentration of organic solvent acetonitrileaffected the protein structure by interfering with the charged partsof the protein.

Figure5: Chromatogram and spectrum of ubiquitin at pH 1.75

Basepeak = 144.9 (1.450e+8=100% pH = 1.75

Thefigure above indicates that at pH 1.75, the high peak is 857.5 at7.283e+7 and varies as the peak decreases.

Figure6: Chromatogram and spectrum of ubiquitin at pH 2.1

Basepeak = 144.9 (1.086e+8=100% pH = 2.1

Thefigure above indicates that relative intensity attained at pH 2.1 wasabout 28% with the high peak being at mz 779.7 at 2.596e+7. Theprotons decreased with an increase in peak values.

Figure7: Chromatogram and spectrum of ubiquitin run at pH of 2.5

Basepeak = 98.8 (9.963e+7=100% pH = 2.5

Peak779.7 high at 1.246e+7, the figure indicates that the peaks fluctuatewith increase in proton and electron charge to mass ratio. This is anindication that pH changes affect the tertiary structure of proteinby interfering with the hydrophobic carboxylic acid side chain.

Figure8: Chromatogram and spectrum of ubiquitin sample run at pH 2.75

Basepeak = 233.1 (8.218e+6=100% pH = 2.75

Thefigure 8 indicates that the peaks did not attain a relative intensityof 100%. The maximum attained relative intensity was 10% indicatingthat there was low mass to charge ratio.

Figure9: Chromatogram and spectrum for ubiquitin sample run at pH 2.75

Basepeak = 233.1 (8.218e+6=100% pH = 2.75

233.1= mass/charge and 383.3 = mass/charge (Equating the two equations

Mass= 233.1(charge) and mass = 383.3 (charge).

Assumingthat the two peaks are related and differ by single charge results inthe equation

m+ 1 = 233.1(z+1) and m = 383.3 (z)

m= (233.1(z+1)-1 and m = 383.3(z)

Assumingthat m is the same for both peaks, the two equations are thus equalto each other

233.1z+232.1 = 383.3 z and z = 2

Massof ubiquitin, therefore, can be determined as

Chargestate calculation unprotonated mass

+1 (383.3-1)*1= 382.3

+2 (383.3-1)*2= 764.6

Averagemass of ubiquitin at pH 2.75 = 573.45

Figure10: Chromatogram and spectrum for ubiquitin sample at pH 7

Chargestate of two peaks = mz

Basepeak = 233.1(1.671e+7= 100%) pH 7

[M+ 8H]8+&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp[M+ 7H]7+&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp[M+ 6H]6+&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp

m/z=&nbsp1508/8&nbsp= 188.5&nbsp&nbsp&nbsp&nbsp&nbspm/z = 1507/7= 215.3&nbsp&nbsp&nbsp&nbsp&nbsp m/z = 1506/6 = 251

[M+ 4H]4+&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp[M+ 2H]2+&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp[M+ H]1+&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp&nbsp

m/z= 1504/4 = 376&nbsp&nbsp&nbsp&nbsp m/z = 1502/2 = 751&nbsp&nbsp&nbsp&nbsp&nbspm/z = 1501/1= 1501

Thefirst assumption that was made in determining the mass was that thetwo adjacent peaks p1 and p2 are from the same compound and alsodiffer by only one charge. Peak value observed in the spectrum equalsmass/charge and the mass = mass of the molecule + protons

233.1= mass/charge and 365.1 = mass/charge (Equating the two equations

Mass= 233.1(charge) and mass = 365.1(charge).

Assumingthat the two peaks are related and differ by single charge, theequation below is generated

m+ 1 = 233.1(z+1) and m = 365.1(z)

m= (233.1(z+1)-1 and m = 365.1(z)

Assumingthat m is the same for both peaks, the two equations are thus equalto each other

233.1z+232.1 = 365.1z and z = 2

Molecularmass of ubiquitin therefore can be determined as

Chargestate calculation unprotonated mass

+1 (365.1-1)*1= 364.1

+2 (233.1-1)*2= 464.2

Calculatedmolecular mass at pH 7 = 414.15


Theresults indicate that when ubiquitin protein was run with organicsolvent 40% methanol, the highest peak achieved was 413.3 at 4.319e+6(Figure 1). The relative intensity was below 5% indicating that theconcentration of methanol affected the protein structure therebylowering the mass to charge ratio. Ions were observed to increase asmass to charge ratio increased. When the same protein was run with20% methanol (Figure 2), the peak was observed to increase to 1224.5at 8.353+6. The relative intensity also rose to 10%. This is anindication that a decrease in the concentration of organic solventsresulted in a reduction effect on the hydrophobic protein component.The same trend was observed when ubiquitin was run with acetonitrileat 20% (Figure 4) and 5% (Figure 3). The relative intensityincreased from 7.5% at 20% acetonitrile to 15% relative intensity at5% acetonitrile. The ions are also increasing with increase in massto charge ratio. This is a clear indication that thethree-dimensional structure of the protein was weakened and the PolarRegions dissolved. Electrons and protons are gained as pH increasesthereby leading to the ionization of the charged particles (Skoog,Holler, and Crouch).

Theresults also indicate that as the pH of the solution increased from1.75 to 7, the charge to mass ratio increased for different samplesof the ubiquitin protein (Figure 5, 6, 7, 8, 9 &amp10). The changeincreased ion concentration in various solutions thereby reducing themass to charge ratio. As a result, the molecular mass of ubiquitinwas affected due to the effect of the charges on the polarity of theproteins. Electrons are distributed at different peaks and increasesas peak increases. As the pH increases, the relative intensity wasalso observed to decrease indicating that ions interactions reducedas pH increased.

Theresults also suggested that the molecular mass of ubiquitin wasobtained as 573.45 at pH of 2.75 (Figure 9). At pH of 7, ifcalculations were done, then the calculated molecular mass ofubiquitin present in the sample would be 414.15 (Figure 10). This isan indication that variation in pH affected the structure ofubiquitin protein hence interfering with mass to charge ratio. Thepeak for the sample run at pH 7 was found to be the smallest (Figure10) compared to the others. The reason for this was as explained. At7, the pH is neutral, and since ubiquitin protein has bothhydrophilic and hydrophobic components in its structure, the proteinis believed to attain a zwitterion structure at this pH. Therefore,the neutral state reduces its charge to mass ratio. As a result, thepeaks that can be formed by the protein at pH 7 are the smallest.

Allthe aims of the experiment were achieved since the high-qualityspectrums were obtained. The molecular mass of ubiquitin was thusdetermined successfully as 573.45. The effects of pH and organicsolvents were also determined and their effects on the structure ofprotein. The pH was observed to affect the concentration of ions andcause a change in mass to charge ratio.


Itcan, therefore, be concluded that the aim of this experiment, whichwas to determine the molecular mass of ubiquitin, was successfullyrealized. The effects of pH on protein suitability were alsodetermined. The experiment established that an increase in pHresulted in a decrease of charge and ionization effect on proteinstructure. Organic solvents were also observed to affect the proteinstructure since they dissolved the hydrophobic components of theprotein amino acid side chain.


&quotLab7: Electrospray Mass Spectrometry.&quot Chemwiki.N.p., 02 Oct. 2013. Web. 18 July 2016.

Murphy,Jennifer. Electrospray Ionization Mass Spectrometry. Chemwiki,n.p.18 Dec. 2013. Web. 18July 2016.

Skoog,Douglas A., F. James Holler, and Stanley R. Crouch. Principlesof Instrumental Analysis.N.P.: n.p., n.d. Print.