ch 3 mass spectrometry

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Ch. 3 Mass Spectrometry

Objectives

To learn the principle of operation of a mass spectrometer

To be able to interpret the mass spectra of atoms and small molecules containing isotopes

To learn how mass spectrometry can be applied to biomolecules such as proteins

To use mass spectrometry to observe the difference between normal adult hemoglobin andsickle cell hemoglobin

To learn the principle of peptide mass mapping to determine the identity of a protein from themass spectrum

Introduction

A mass spectrometer is an instrument that is used to measure atomic and molecular mass

directly. This instrument was developed in the 1920s and has been one of the most widely used

analytical techniques because of its versatility. A mass spectrometer is capable of providing

information about isotopic ratios of atoms in samples, qualitative and quantitative composition of

inorganic and organic analytes in complex mixtures, and structures in a wide variety of

molecules. Recent discoveries in the field of mass spectrometry have also enabled the analysis

of large biomolecules, such as proteins.

The major components of any mass spectrometer are shown in Figure 1. The purpose of the

inlet system is to introduce a very small amount of sample (typically < 1 micromole) into the

instrument and convert the sample to the gaseous phase. The ion source of the mass

spectrometer converts the components of the sample into ions by bombardment with electrons,

ions, molecules, or photons. The mass analyzer functions to disperse the ions based on mass to

charge ratio and the detector quantifies the amount of ion with a given mass to charge ratio by

measuring a current.

Inlet System Ion SourceMass

AnalyzerDetector

Vacuum (10-5 10-8 torr)


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Figure 1: Simplified components of a mass spectrometer.

There are several different types of mass spectrometers, but in this experiment we will

describe only two different designs. The first is a simple design based on the deflection of ions

in a magnetic field. Figure 2 shows a schematic of this instrument. The sample is introduced

into the instrument and vaporized (by heating) if it is not already in the gaseous state. It is then

ionized by collisions with high-energy electrons produced from a heated filament. Collisions

with the electrons result in positively charged ions of the analyte (A).

A (g) + e- A

+(g) + 2e

-

Electron impact ionization is carried out with electron energies that are high enough to break

the covalent bonds within a molecule, resulting in molecular fragments. Fragmentation is often

useful in deducing the structure of the parent molecule. All the ions are then moved through the

mass spectrometer by an electrostatic potential. They are focused into a narrow beam before

passing through a magnetic field, which deflects the ions by varying amounts depending on their

mass to charge ratio. Since most of the analyte atoms or molecules acquire a +1 charge in the

ionization process, the end result is that the ions are separated spatially by mass. As each

positive ion strikes the detector, a burst of electrons are ejected, which is measured by

amplification.


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Figure 2: Schematic of a magnetic sector mass spectrometer utilizing electron impact as an

ionization source. (Moore, Stanitski, Jurs, Chemistry, The Molecular Science, 1st Ed., pg. 54,

Brooks/Cole, 2003).

The results are plotted as a mass spectrum which is a graph that shows the ion abundance

versus the mass of the ions. The mass spectrum for the element Neon is shown in Figure 3. Each

isotope of Ne produces a different peak in the mass spectrum, and the height of the peak is

proportional to the number of atoms of each isotope present. Therefore, a mass spectrometer can

be used to obtain a complete analysis of isotopic composition of an element. This type of mass

spectrometer was used to generate the mass spectrum you will analyze for Hg, Cl2, and CH2Cl2.

Figure 3: Mass spectrum for Neon generated by a magnetic sector instrument.

The second type of mass spectrometer we will describe is used for the analysis of large

biomolecules. Masses in the range 1000 500,000 Daltons (1 Da = 1 amu) can be routinely

analyzed. This type of instrument was used to acquire the mass spectra for normal adult

hemoglobin, sickle cell hemoglobin, and protein that you will identify using peptide mass

mapping. The two main differences between this type of instrument and the magnetic sector

instrument described previously are in the vaporization/ionization method and the mass analyzer.

Matrix assisted laser desorption ionization (MALDI) is the method used for both vaporization

and ionization of the solid biomolecules. This method incorporates the biomolecule into a

specifically chosen matrix of smaller organic molecules. The matrix, along with the

0

10

20

30

40

50

60

70

80

90

100

18 19 20 21 22 23

Mass Number

Intensity

90.5%

0.3%9.2%


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biomolecules, are vaporized and ionized by a laser pulse that heats the mixture. The evaporative

cooling of the matrix allows the biomolecule to remain intact in the transition to the gas phase.

The mass analyzer is called a Time of Flight analyzer (TOF) and it separates masses without a

magnetic field. It works by taking advantage of the differing velocities of ions accelerated

through the same voltage. The physics of the separation is straightforward. If ions of different

mass are accelerated through the potential and then allowed to pass through an evacuated tube,

they will not arrive at the opposite end at the same time. Figure 4 shows a diagram of the Time

of Flight analyzer. In this type of analyzer, protein ion flight times are typically tens of

microseconds, but adjacent masses reach the detector separated in time only by nanoseconds.

sample gas Detector

Ionizer

Figure 4: Diagram showing the separation of analytes of different masses using a time of

flight mass spectrometer. The ionized molecules or atoms are drawn and accelerated into an

evacuated tube by pulsing a voltage. The ions, which usually acquire a charge of +1, have

different masses and separate as they pass through the evacuated tube. The detector must operate

at a high rate so as to detect the individual masses arriving with nanosecond time resolution.

Procedure: Mass Spectral Analysis

Section 1: Isotopic Abundance of Mercury

Figure 5 shows the mass spectrum for elemental mercury. Six intense mass spectral peaks

can be observed corresponding to masses of 198, 199, 200, 201, 202, 204. In addition, there is a

small peak at mass 196 attributable to the seventh stable isotope of mercury. Many of the other

small peaks cannot be attributed to any particular species. Table 1 shows the measured mass for

each peak, the absolute intensity, and the relative intensity as compared to the most intense peak

(called the base peak and assigned an intensity of 100).


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Figure 5: Mass Spectrum for Elemental Mercury

Table 1: Mass Spectral Data for Elemental Mercury obtained on the Kratos MS80

system in the Mass Spectrometry Laboratory at Indiana University.


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Table 2: Exact Masses and Fractional Abundances for Mercury

Questions about Hg

1) Use the absolute intensity for each of the seven stable isotopes to calculate an observed

relative abundance in % for each of the seven isotopes from the experimental data in Table 1.

2) Use the % abundance calculated in #1 and the experimentally measured mass to calculate

an observed average atomic mass.

3) Use Table 2 containing exact masses and fractional abundances for mercury to calculate a

theoretical average atomic mass.

4) Calculate the % difference between the observed and theoretical atomic masses for

mercury.

5) Provide a reasonable guess to the identity of the peak at 207.07 amu that is not due to an

isotope of Hg.


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Section II: Isotopes and Fragmentation in Molecules

A. Chlorine

The mass spectrum for chlorine is shown in Figure 6. There are peaks in two regions

corresponding to masses between 34 and 40 and 68 and 75.

Figure 6: Mass spectrum for chlorine (Cl2).

Questions about Cl2

1) What isotopes of Cl are observed? Explain your reasoning.

2) For each peak in the mass spectrum identify the list all the positive ions that might

contribute to the peak.

3) Use the relative intensity of the peaks in the mass spectrum to calculate the fractionalabundance of the most abundant isotope of Cl to the fractional abundance of the least abundant

isotope of Cl.

4) Compare your answer in #3 to known values.


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B. Methylene chloride (CH2Cl2)

Figure 7: Mass Spectrum for Methylene Chloride

Table 3: Mass Spectral Data for Methylene Chloride (CH2Cl2)


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Table 4: Exact Masses and Fractional Abundance for Carbon, Hydrogen, and Chlorine

Mass Number Exact Mass Abundance

12 (C) 12.000000 0.9890

13 (C) 13.003354 0.0110

1 (H) 1.007825 0.9998

2(H) 2.014102 0.0002

35 (Cl) 34.968851 0.7577

37 (Cl) 36.965898 0.2423

Questions about Methylene Chloride:

1) Refer to Table 4: Exact Masses and Fractional Abundance for Carbon, Hydrogen, and

Chlorine and predict which isotopes of C, H, and Cl can be ignored in the analysis of the mass

spectrum.

2) For each of the 8 peaks in the mass range 80-90, make a list of the + ions that might give

rise to the peak.

3) Rationalize in quantitative terms the intensities of these peaks by referring to the isotopic

abundances for C, H, and Cl.

Section III: Mass Spectrometry of Proteins

A. MALDI-TOF Analysis of Intact Hemoglobin

Figure 8 shows the MALDI-TOF Mass spectrum of normal adult hemoglobin and adult

sickle cell hemoglobin. Sickle cell hemoglobin results from a mutation in a single gene that

causes one amino acid to be changed on the chain of hemoglobin. The mass difference

between the normal and sickle cell chains shown below is 30.30.8 Da. This spectrum

demonstrates that MALDI-TOF is useful technique in screening hemoglobin for abnormalities.

This is important because many states (almost 40) in the US require newborn blood screening for

early detection of hemoglobin-related diseases. MALDI-TOF offers advantages over the more

commonly used screening technique of electrophoretic analysis because it has faster analysis

times and good resolution.


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Figure 8: MALDI-TOF Analysis of Normal Adult Human Hemoglobin and Sickle Cell

Hemoglobin. The measured mass difference between the two chain peaks = 30.30.8 Da.

Questions

1) Given a table of amino acids give possible amino acid substitutions that could account for

the mass difference in the Beta chain of the sickle cell hemoglobin. (Given on last page of this

experiment)

2) Use various library and web resources to search for the actual amino acid substitution that

occurs. Give the source where you found this information.

B. Use of Peptide Mass Mapping to Determine the Identity of an Unknown Protein

The mass spectrometric analysis of proteins is a rapidly developing field that will most likely

make a large contribution in our understanding various diseases (such as cancer) at the molecular

level. This understanding is the first step in the development of drugs to treat the disease. One


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approach to understanding the difference between a healthy and diseased cell is to study the

genetic sequence in DNA. Although proteins are coded by DNA, the majority of proteins

undergo post-translational modification. The modifications could be the addition of sugar

groups catalyzed by enzymes, phosphorylated sites, or binding of essential metals.

Phosphorylation, for example, is often used to regulate enzyme activity. Scientists are interested

in determining the differences in the proteins that are expressed between healthy and diseased

tissue or cells. There are two main steps in this analysis. The first is the separation and

visualization of proteins using 2D gel electrophoresis. Prospective disease-related proteins can

be recognized by comparing gel patterns of proteins derived form healthy and diseased tissue.

Once the individual protein spot is targeted for further analysis, it is extracted and digested with

a protein called trypsin. Trypsin cleaves the protein at lysine (K) and arginine (R) residues, thus

breaking the protein into smaller fragments.

Parent Protein

GASEMHKYWINLCTYDQRVPSAGTCFHHEQD

Fragments after Tryptic Digest:

GASEMHK

YWINLCTYDQR

VPSAGTCFHHEQD

Figure 9 shows the MALDI-TOF mass spectrum of a tryptic digest of an unknown protein

and Table 5 gives a list of peaks that are above threshold intensity. Your task is to use the mass

spectrum of the tryptic digest of the unknown protein and a protein mass spec data base available

on the web to perform a technique called peptide mass mapping, which will enable you

determine the identity of the protein.

Many forces have come together in the last decade allowing mass spectrometry to become

one of the central tools in biology and biochemistry. Among these are the proliferation of

protein sequence databases, the publication of numerous complete organism genomes, the

development of mass spectrometers capable of providing accurate masses for large biological

molecules, and a vast increase in affordable computer processing power. Peptide mass mapping

is one of these new mass spectrometric techniques. In peptide mass mapping, an unknown


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protein is digested with an enzyme, and the mass spectrum of the resulting protein fragments is

recorded. The observed masses are compared against a list of all protein fragments that could be

present in the sample (based on knowledge of the translated genome and the cleavage chemistry

of the enzyme). At its simplest level, the protein with the most predicted fragment masses

matching observed fragment masses is though to be present in the sample. No single match

between an observed mass and theoretical mass identifies a protein. Rather, it is the series of

matches that allows an identification to be made.

Figure 9: Mass Spectrum of Unknown Protein


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Table 5: List of Peaks above threshold level in Unknown Protein

Peak Mass (Da) Rel Int Abs Int Point

---- --------------- ------- ---------- ----------

1 861.045 0.0330 2.0700e+02 6701.35

2 877.007 0.0293 1.8400e+02 7065.38

3 932.471 0.0296 1.8600e+02 8305.20

4 952.490 0.0793 4.9800e+02 8743.63

5 1071.578 0.1156 7.2600e+02 11261.43

6 1087.656 0.0973 6.1100e+02 11590.39

7 1126.595 0.0239 1.5000e+02 12377.20

8 1149.721 0.0204 1.2800e+02 12838.06

9 1161.660 0.0250 1.5700e+02 13074.17

10 1165.602 0.0201 1.2600e+02 13151.86

11 1171.719 0.0848 5.3300e+02 13272.16

12 1274.754 0.5713 3.5890e+03 15253.16

13 1314.690 0.3489 2.1920e+03 15999.37

14 1378.655 0.0415 2.6100e+02 17171.30

15 1449.813 0.2139 1.3440e+03 18443.52

16 1471.789 0.0229 1.4400e+02 18830.08

17 1529.785 1.0000 6.2820e+03 19836.60

18 1669.917 0.0478 3.0000e+02 22192.55

19 1833.896 0.2486 1.5620e+03 24827.14

20 2058.955 0.1315 8.2600e+02 28258.15

21 2074.927 0.0209 1.3100e+02 28494.34

22 2080.930 0.0263 1.6500e+02 28582.87

23 2213.107 0.0519 3.2600e+02 30500.89

24 2228.187 0.0519 3.2600e+02 30716.02

25 2341.184 0.0642 4.0300e+02 32305.49

26 2996.464 0.0441 2.7700e+02 40849.31

27 3124.524 0.0154 9.7000e+01 42405.65

28 3265.605 0.0153 9.6000e+01 44083.76


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Instructions for Identifying the Protein

1) Load the following protein prospector web site. http://prospector.ucsf.edu/

2) Under Sequence Database Search Programs select MS-Fit.

3) Under Data Base choose SwissProt 2005.01.06

4) Digest = Trypsin

5) Maximum number of missed cleavage = 2.

6) Cys modified by = unmodified

7) Possible modification = oxidation of M

8) Sample ID = Magic Bullet Digest

9) Minimum matches = 4

10) Maximum reported hits = 10 (this one is important)

11) Sort By = Score sort

12) In the data paste area, type in the masses of the peaks in table 5.

13) Mass tolerance = 0.1 Da

13) Go to the middle of the page and select start search.

Questions

1) In the results summary section, write down the protein name and the organism for the top

10 matches. The name for the organism can be found by clicking on Accession # and reading

the 8th

line down (OS for organism source).

2) You have determined that the mass spectrum was a tryptic digest of human hemoglobin.

Next generate a % coverage map for the and chain of human hemoglobin. To do this you

will compare the number of amino acids that were found in the mass spectral data with the

number of amino acids in the overall sequence.

a) The amino acid sequence for the chain of human hemoglobin is given below. It is

broken down into sequences of 10 AAs for easy viewing.

VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQR FFESFGDLST PDAVMGNPKV

KAHGKKVLGA FSDGLAHLDN LKGTFATLSE LHCDKLHVDP ENFRLLGNVL VCVLAHHFGK

EFTPPVQAAY QKVVAGVANA LAHKYH
http://prospector.ucsf.edu/http://prospector.ucsf.edu/http://prospector.ucsf.edu/


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In the detailed results section for the chain of human hemoglobin match each of the

database sequence peptides (given in blue) with the whole sequence of the protein. In other

words, for each peptide found, highlight this region in the whole sequence. (Hint: The # of the

amino acid where the peptide starts is given.) To calculate a % coverage divide the number of

amino acids found by the total number of amino acids and multiply by 100.

b) Generate a % coverage map for the chain of human hemoglobin. The entire AA

sequence is given below.

VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSFPTTK TYFPHFDLSH GSAQVKGHGK

KVADALTNAV AHVDDMPNAL SALSDLHAHK LRVDPVNFKL LSHCLLVTLA AHLPAEFTPA

VHASLDKFLA SVSTVLTSKY R

c) Can you think of any reasons why the coverage is not 100%? (extra credit)

3) Compare the AA sequence for human hemoglobin to other species hemoglobin given

below.

Gorilla

VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQR FFESFGDLST PDAVMGNPKV

KAHGKKVLGA FSDGLAHLDN LKGTFATLSE LHCDKLHVDP ENFKLLGNVL VCVLAHHFGK

EFTPPVQAAY QKVVAGVANA LAHKYH

Ring Tailed Coati (the raccoon of Central America)

VHLTGEEKTA VTNLWAKVNV DEVGGEALGR LLVVYPWTQR FFESFGDLSS PDAIMGNPKV

KAHGKKVLNS FSEGLKNLDN LKGTFAKLSE LHCDKLHVDP ENFRLLGNVL VCVLAHHFGK

EFTPQVQAAY QKVVAGVANA LAHKYH

Are there differences? If so, how many?

Is there a major difference in the amino acid structure of these substitutions? Use your table

of amino acids attached at the end to comment on this.


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Acknowledgements

1) The material and questions from the mass spectrometric analysis of Hg, Cl 2, and CH2Cl2

were adapted from

Peters, Dennis, S125 Laboratory Manual for S125, pg. 10-36, Tichenor Publishing,

Bloomington, IN, 2002.

2) Protein data were obtained from research group of Professor Jim Reilly, Indiana

University.

3) A special thanks to John Karty for an explanation of peptide mass mapping and assistance

in writing questions.

ch 3 mass spectrometry

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