This document provides an overview of protein mass spectrometry data analysis. It describes the key components of a mass spectrometer, including the ion source, mass analyzer, and detector. It discusses common ionization techniques like ESI and MALDI and mass analysis methods like quadrupole and time-of-flight. The document then covers the process of analyzing mass spectrometry data, including identifying proteins and peptides from spectra. It presents a case study on analyzing depleted and non-depleted blood plasma samples. Finally, it lists several applications of mass spectrometry like proteomics, disease biomarker detection, and pharmaceutical analysis.

1. Presented by:
Nahid Rehman(2020RBT03)
Reena Joshi(2020BT08)
PROTEIN MASS SPECTROMETRY
DATA ANALYSIS

2. CONTENTS:
• INTRODUCTION
• COMPONENTS OF A MASS SPECTROMETER
ION SOURCE
ANALYZER
DETECTOR
• MASS SPECTROMETRY DATA ANALYSIS
• CASE STUDY
• APPLICATIONS OF MASS SPECTROMETRY.

3. INTRODUCTION:
• Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge
ratio of ions.
• The results are typically presented as a mass spectrum, a plot of intensity as a function
of the mass-to-charge ratio.
• Mass spectrometry is used in many different fields and is applied to pure samples as
well as complex mixtures.
• The molecules in a test sample are converted to gaseous ions that are subsequently
separated in a mass spectrometer according to their mass-to-charge (m/z) ratio and
detected.

4. • Main information given in MS analysis:
1. molecular weight
2. number of specific elements (based on isotope peaks)
3. molecular formula (with high resolution MS)
4. reproducible fragment patterns (to get clues about functional groups and/or arrangement of
components or to confirm compound identity)
• Protein mass spectrometry refers to the application of mass spectrometry to the study
of proteins.
• Its applications include :-
1. identification of proteins and their post-translational modifications,
2. the elucidation of protein complexes,
3. their subunits and functional interactions,
4. used to localize proteins to the various organelles.

5. COMPONENTS OF A MASS SPECTROMETER

6. 1.Sample inlet:
This comprises a sample or target plate; a high-performance liquid chromatography
(HPLC), gas chromatography (GC) or capillary electrophoresis system; solids probe;
direct chemical ionisation chamber.
2.ION SOURCE:
• The ion source is the part of the mass spectrometer that ionizes the material
under analysis (the analyte). The ions are then transported by magnetic or electric
fields to the mass analyzer.
• Ions may be produced from a neutral molecule by removing an electron to
produce a positively charged cation, or by adding an electron to form an anion.
• Hard ionization techniques are processes which impart high quantities of residual
energy in the subject molecule invoking large degrees of fragmentation.
• Resultant ions tend to have m/z lower than the molecular mass.
• The most common example of hard ionization is electron ionization (EI).

7. • Soft ionization refers to the processes which impart little residual energy
onto the subject molecule and as such result in little fragmentation.
Examples include:-
 Fast atom bombardment (FAB)
 Chemical ionization (CI)
 Atmospheric-pressure chemical ionization (APCI)
 Electrospray ionization (ESI)
 Desorption electrospray ionization (DESI)
 Matrix-assisted laser desorption/ionization (MALDI)
• Proteins and peptides are usually ionized via protonation in a spectrometer
because of their NH2 groups that accept a H1 ion. .
• In proteomics analysis, the most commonly used two devices for ionization are
MALDI and ESI. Both of them are soft ionization techniques, and ions undergo
little fragmentation.

8. Electrospray Ionisation(ESI)
• ESI uses electrical energy to assist the transfer of ions from solution into the gaseous phase before they are subjected to mass
spectrometric analysis.
• The transfer of ionic species from solution into the gas phase by ESI involves three steps:
(1) dispersal of a fine spray of charge droplets, followed by
(2) solvent evaporation and
(3) ion ejection from the highly charged droplets tube, which is maintained at a high voltage (e.g. 2.5 – 6.0 kV) relative to the
wall of the surrounding chamber.

9. • A mist of highly charged droplets with the same polarity as the capillary voltage
is generated. The application of a nebulising gas (e.g. nitrogen), which shears
around the eluted sample solution, enhances a higher sample flow rate.
• The charged droplets, generated at the exit of the electrospray tip, pass down a
pressure gradient and potential gradient toward the analyser region of the mass
spectrometer.
• With the aid of an elevated ESI-source temperature and/or another stream of
nitrogen drying gas, the charged droplets are continuously reduced in size by
evaporation of the solvent, leading to an increase of surface charge density and a
decrease of the droplet radius.
• Finally, the electric field strength within the charged droplet reaches a critical
point at which it is kinetically and energetically possible for ions at the surface of
the droplets to be ejected into the gaseous phase.
• The emitted ions are sampled by a sampling skimmer cone and are then
accelerated into the mass analyser for subsequent analysis of molecular mass and
measurement of ion intensity.

10. Matrix-assisted laser desorption ionization (MALDI)
• MALDI is basically a laser desorption ionization method wherein ionization is assisted
by small organic molecules, called matrix. The matrices used in positive ion mode
MALDI mass spectrometry are organic acids that have strong absorption for the
wavelength of the laser used.

11. • The sample for MALDI is usually prepared in one of the following ways:
1.mixing the analyte solution with the matrix solution → deposition of the mixture on a
metallic plate → complete drying of the sample
2.Deposition of the matrix on the metallic plate → drying of matrix → addition of analyte
solution → drying of analyte solution
• Desorption and ionization is achieved by applying the laser pulse on the dried sample.
It is believed that the absorption of light by the matrix molecules causes sublimation of
matrix crystals carrying along with them the analyte molecules into the gas phase .
• MALDI can very efficiently generate the gas phase ions from a variety of non-volatile
and thermolabile molecules such as proteins, carbohydrates, nucleic acids, and
synthetic polymers.
• MALDI can desorb and ionize the molecules as large as 300 kDa. MALDI usually results
in the molecular species having only one charge.

12. 3.MASS ANALYZER
• After ionization, the ionized molecules of peptides or proteins enter the mass
analyzer section of spectrometer.
• In the mass analyzer, molecules are separated based on their mass-to-charge
ratio by electric and/or magnetic fields or by measuring the time an ion takes to
reach a fixed distance from the point of ionization to the detector.
• For the separation of ionized molecules, different kinds of mass analyzers are
available such as Quadrupoles, Time-of-flight (TOF), Magnetic sectors, Fourier
transform, and Quadrupole ion traps.
• In proteomics, quadrupole and the TOF analyzers are mostly used.
• MS with the ESI device usually carries a quadrupole analyzer and MALDI ion
source is often used with TOF analyzers.

13. Time-of-flight.
• A TOF spectrometer separates ions based on their velocity and can, essentially, be thought of as a race
from a starting point to the detector.
• Theoretically, the ions are all formed at the same time and place in the ion source and then accelerated
through a fixed potential (for example, 1–20 kV) into the TOF drift tube.
• After the ions are accelerated they travel through a fixed distance, typically 0.5–2.0 metres, before
striking the detector. Thus, by measuring the time it takes to reach the detector after the ion is formed,
the m/z of the ion can be determined.
• As all the ions with same charge obtain the same KINETIC ENERGY after acceleration, the lower m/z ions
achieve higher velocities than the higher m/z ions.
• Ion velocities are inversely related to the square root of m/z.

14. 4.DETECTOR
• The detector is the final component of a mass spectrometer and is used for monitoring
and recording the presence of separated ions coming from the mass analyzer.
• Depending on the analytical applications and design of the instrument, different
detectors can be used such as -
 electron multiplier
 Faraday cup
 negative-ion detection
 postacceleration detector
 channel electron multiplier array
 photomultiplier conversion dynode
 array detector.
• After detection of ions, the signals are recorded on a graph by plotting the amount of
signal versus m/z ratio.

15. Photomultiplier (or Scintillation Counter)
• In a photomultiplier (or scintillation counter) the ions initially strike a dynode which results in electron
emission. These electrons then strike a phosphorous screen which in turn releases a burst of
photons.
• Photons then pass into the multiplier where amplification occurs in a cascade fashion - much like
with the electron multiplier.
• The main advantage of using photons is that the multiplier can be kept sealed in a vacuum
preventing contamination and greatly extending the lifetime of the detector.

16. MASS SPECTROMETER DATA
ANALYSIS

17. MASS SPECTROMETER DATA ANALYSIS
A mass spectrum of the fragment ions, known as a tandem mass (MS/MS) spectrum, is obtained
for each selected precursor. All the mass spectra are written to a file or loaded into a database
and are then further analyzed, using the informatics techniques described to identify the peptides
and proteins present in the sample.
 Resource for finding software tools for proteomics is the www.proteomecommons.org web site.
 proteomics experimental data usually involve most or all of these steps:
conversion to and processing via open data formats
spectrum identification with a search engine
validation of putative identifications
protein inference
quantification
organization in local data management systems
interpretation of the protein lists
transfer to public data repositories.

18. The majority of protein sequence analysis today
uses mass spectrometry. There are several steps in
analyzing a protein.
Digest the protein to peptides (in gel or solution). Mass spectrometry currently gets limited sequence data from whole
proteins, but can easily analyze peptides.
Trypsin is first choice for digestion-readily available, specific,
After digesting a protein with trypsin or some other specific proteinase, the masses of the intact peptides are measured,
usually with a MALDI instrument. A program compares the observed masses of peptides with those calculated from the
digestion of all proteins in a database.
Separate peptides, usually on reverse phase column with acetonitrile gradient. We use columns 75 µm in diameter. We
use acetic acid in the solvents because the commonly used tri-fluoro acetic interferes with ionization.
Place ionized peptides in vapor phase by passing the column eluate, containing peptides and solvent, through a fine tip
to form tiny droplets. After evaporation of solvent, peptides are left in the vapor phase. Charged surfaces move the
ionized peptide into the mass spectrometer. LC-MS (liquid chromatography mass spectrometry or HPLC-MS
chromatography) is used to introduce molecules into a mass spectrometer

19. Measure mass of peptides.
Fragment peptides. Collisions with gas molecules fragments peptides at peptide
bonds.
Measure mass of fragments from peptides. Because there are two steps of mass
spectrometry (mass of peptide, mass of fragment of peptide), this is called
MS/MS, or MSn because there can be 2 or more fragmentation steps. A two step
process is also called tandem mass spectrometry.
Use fragment mass data to determine the sequence of the peptide by seeing
which combinations of amino acids gives the observed masses of peptide
fragments

20. CASE STUDY: Such-Sanmartín et al (2014)
About KYSS
• ‘‘Know Your Samples’’ (KYSS) for assessment and visualisation of largescale
proteomics datasets, obtained by mass spectrometry (MS) experiments.
• KYSS facilitates the evaluation of sample preparation protocols, LC peptide
separation, and MS and MS/MS performance by monitoring the number of
missed cleavages, precursor ion charge states, number of protein
identifications and peptide mass error in experiments. KYSS generates
several different protein profiles based on protein abundances, and allows
for comparative analysis of multiple experiments.
• KYSS was adapted for blood plasma proteomics and provides
concentrations of identified plasma proteins.
• The KYSS software is open source and is freely available at
http://kyssproject.github.io/.

21. Cont..
Proteome analysis of blood plasma samples
• Blood plasma samples were processed with or without prior depletion of
abundant proteins based on hydrogel particles protein depletion.
• Following trypsin digestion, peptide mixtures were analysed by LC–MS/MS using
an Easy-nLC II (Thermo Fisher Scientific, Odense, Denmark) coupled to a LTQ
Orbitrap XL hybrid instrument (Thermo Fisher Scientific, Bremen, Germany).
• Protein identification was performed with Proteome Discoverer v1.4.0.270
(Thermo Fisher Scientific) and Mascot v2.3.02.
• Peak areas were calculated based on the top-3/Hi3 precursor area quantification
method.
• Information. Raw data files and text files including lists of protein / peptide
identifications obtained by sequence database searching are available in the
ProteomicsDB repository
(https://www.proteomicsdb.org/proteomicsdb/#projects/4112).
• The files that contain the lists of protein and peptide identifications are in a
format that can be directly submitted to KYSS.

22. Cont..
RESULTS & DISCUSSION
• KYSS software was developed to facilitate detailed evaluation of large scale proteome
datasets obtained by LC–MS/MS by taking advantage of the information contained in
reports generated by sequence database searching tools.
• human blood plasma samples Was analysed by LC–MS/MS and subsequently searched
the MS/MS spectra using Mascot and Proteome Discoverer.
• The resulting protein/peptide list was exported as plain-text data files and submitted to
KYSS.
• KYSS was utilised to
(1) assess the performance of different proteomics workflows,
(2) visualise the physico-chemical properties of identified proteins, and
(3) demonstrate the efficiency of hydrogel particles in depleting the abundant proteins in
plasma.

24. Applications
Mass spectrometry has both qualitative and quantitative uses which include identifying unknown
compounds, determining the isotopic composition of elements in a molecule, and determining
the structure of a compound by observing its fragmentation.
 Quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion
chemistry.
 Trace gas analysis, Atom probe
 Pharmaceutical (drug discovery)- used to identify the mixtures of complicated samples such as urine,
lymph, blood, and it is used with high sensitivity methods to measure low doses and long time point data.
 Characterization of Protein ( electrospray ionization (ESI) and matrix-assisted laser
desorption/ionization (MALDI) are the two primary methods for ionization of whole proteins) Protein
Identification, Protein sequencing, Bacterial Identification
Disease Biomarker detection.
To determine the structure of a compounds by observing its fragmentation
It is used in quantifying the compound's amount in a sample.

26. References.
• Such-Sanmartín et al (2014) Mass spectrometry data quality assessment for protein analysis and large-scale proteomics
http://dx.doi.org/10.1016/j.bbrc.2014.01.066
• Eric W. Deutsch et al (2018) Data analysis and bioinformatics tools for tandem mass spectrometry in proteomics.
doi:10.1152/physiolgenomics.00298.2007
• Sparkman, O. David (2000). Mass spectrometry desk reference. Pittsburgh: Global View Pub. ISBN 978-0-9660813-2-9.
• Principles and Techniques of Biochemistry and Molecular Biology Seventh edition EDITED BY KEITH WILSON AND JOHN WALKER
• Rauniyar, Navin & Stevens, Jr, Stanley & Prokai, Laszlo. (2007). Fourier transform ion cyclotron resonance mass spectrometry of
covalent adducts of proteins and 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation. Analytical and bioanalytical
chemistry. 389. 1421-8. 10.1007/s00216-007-1534-2.
• Ho, C. S., Lam, C. W., Chan, M. H., Cheung, R. C., Law, L. K., Lit, L. C., Ng, K. F., Suen, M. W., & Tai, H. L. (2003). Electrospray
ionisation mass spectrometry: principles and clinical applications. The Clinical biochemist. Reviews, 24(1), 3–12.
• NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics
• Glish, G., Vachet, R. The basics of mass spectrometry in the twenty-first century. Nat Rev Drug Discov 2, 140–150 (2003).
https://doi.org/10.1038/nrd1011.
• Büyükköroğlu, G., Dora, D. D., Özdemir, F., & Hızel, C. (2018). Techniques for Protein Analysis. Omics Technologies and Bio-
Engineering, 317–351. doi:10.1016/b978-0-12-804659-3.00015-4 .
• Stauffer, E., Dolan, J. A., & Newman, R. (2008). Gas Chromatography and Gas Chromatography—Mass Spectrometry. Fire Debris
Analysis, 235–293. doi:10.1016/b978-012663971-1.50012-9

27. Submitted To:
Dr. Ashutosh Mani
MNNIT