1. MS Data analysis for Proteomics studies
The process of inferring accurate protein identification data from thousands of mass spectra generated in mass spectrometry based proteomics experiments is a complicated and challenging process. Improved computation and greater data storage capability developed over the last decade has now considerably simplified this process.
Title of the concept
Suruchi Rao
Harini Chandra

2. Proteolysis (trypsin digestion)
Master Layout (Part 1) 1
This animation consists of 3 parts:
Part 1 – Typical proteomics experiment
Part 2 – Peptide Mass Fingerprinting (PMF)
Part 3 – MS/MS Data analysis
SDS-PAGE
2-DE 2
Proteolysis (trypsin digestion) 3
Tandem MS/MS
MALDI + 4
Mass spectra 5

3. Definitions of the components: Part 1 – Typical proteomics experiment
1. Typical Proteomics Experiment: One that involves the use of a Mass Spectrometer to analyze the content of a proteome or to elucidate individual components of a protein complex after they have been suitably separated by various gel-based or chromatographic techniques.
2. SDS-PAGE: SDS-PAGE is a separation technique that brings about protein separation under denaturing conditions. This is extensively used along with quantitative proteomics techniques like iTRAQ, SILAC etc. Once the proteins have been separated, the gel can be cut into pieces and the desired bands can be eluted out, which can then be taken for further identification by MS.
3. 2-DE: The commonly used protein separation technique that carries out fractionation of the protein mixture based on isoelectric point in one dimension and molecular weight in the second dimension. Protein bands from the gel can be excised and eluted using a suitable buffer and used for further analysis by MS.
4. Proteolysis: The process of site-specific digestion of proteins, typically by the proteolytic enzyme, Trypsin, which generates peptide fragments of appropriate size that are analyzed in the form of positive ions in MS. 2 3 4 5

4. Definitions of the components: Part 1 – Typical proteomics experiment
5. Tandem MS/MS: This is a MS technique that makes use of a combination of ion source and two mass analyzers, separated by a collision cell, in order to provide improved resolution of the fragment ions. The mass analyzers may either be the same or different. The first mass analyzer selects only a particular ion which is further fragmented and resolved in the second analyzer. This can be used for protein sequencing studies. 6. Matrix Assisted Laser Desorption Ionization (MALDI): MALDI is an efficient process for generating gas-phase ion of peptides and proteins for mass spectrometric detection. Target plate with dried matrix-protein sample is exposed to short, intense pulses from a UV laser. 7. Mass spectra: Charged peptide fragments are resolved by the mass analyzer on the basis of their mass-to-charge ratios and then detected by means of the detector, which generates a spectrum of relative abundances of the ions against their mass-to-charge ratio. 2 3 4 5

5. Tube containing trypsin & buffer
Part 1, Step 1 1
Proteolytic digestion 2
SDS-PAGE
2-DE
Trypsin 3
Peptide fragments
Protein of interest 4
Tube containing trypsin & buffer
Action
Description of the action
Audio Narration
First show the two squares on top with the black patterns on them. Then show the red circle followed by the tube below & the two arrows. The black dots in the circle must enter the tube. This must then be zoomed into and the violet shape in the box must be shown. The green object must then appear which must move along the violet shape breaking it up into small fragments (shown on the right) as it moves.
Most proteomics experiments involve the separation of a protein mixture by means of electrophoresis followed by elution of the protein band of interest. This protein is then digested into small peptide fragments by means of proteolytic enzymes, the most commonly used one being trypsin. These small peptide fragments can then be further analyzed by MS
As shown in animation. 5

6. Spectra of analyte protein
Part 1, Step 2 1
Mass Spectrometry analysis – MALDI TOF
Spectra of analyte protein
Tryptic digest 2
Laser source
Applied to sample plate
Detector 3 + + +
TOF tube
MALDI Sample plate 4
Reflector
Action
Description of the action
Audio Narration
First show the tube marked ‘tryptic digest’ followed by the down arrow with label and the setup shown below that. Next show a light coming out of the red cylinder which must hit the white plate on the left and then move towards the white ‘reflector’ on the right end of the tube and finally must be deflected onto the detector. Next show the ions of different sizes appearing which must move at different speeds across the tube with the smallest ones moving the fastest and largest moving slowly. They must move until they reach the detector after which the graph above must be shown.
The peptide fragments obtained after digestion can be analyzed either by MALDI-TOF or by Tandem MS/MS. In MALDI-TOF, peptide ions are accelerated at different velocities depending on their mass to charge ratios. The spectrum generated provides a set of peaks whose masses represent each of the peptides present in the mixture. These spectra can then be analyzed by various available softwares to obtain more information about the protein.
As shown in animation. 5

7. Spectra of analyte protein
Part 1, Step 3 1
Mass Spectrometry analysis – Tandem MS/MS
Tryptic digest 2
Detector
Spectra of analyte protein
Peptide ions generated
Ions of selected m/z 3 + +
Q2 – Collision cell
Q3 – RF mode
Peptide ions
Q1 – Scanning mode
Fragmented ions 4
Audio Narration
Action
Description of the action
First show the tube on top marked ‘tryptic digest’ followed by the down arrow with label followed by the coloured ions and the remaining components. The ions must move towards the first set of rods & only the pink ions must be allowed through the opening. These must enter the orange cube. In this, they must get fragmented into smaller pieces and must come out of the other end as shown. These smaller pieces must fly through the second set of rods and enter the detector. As each of the fragments reaches the detector, the graph on the right must start appearing from left to right until all the fragments have been detected.
Tandem MS/MS is capable of providing more in-depth sequence information. Each peptide in the digest is further fragmented in the second ionization step and analyzed, thereby generating a spectrum for each peptide. These spectra can then be analyzed by various available softwares to obtain more information about the protein.
As shown in animation. 5

8. 1 Master Layout (Part 2) 2 3 4 5 This animation consists of 3 parts:
Part 1 – Typical proteomics experiment
Part 2 – Peptide Mass Fingerprinting (PMF)
Part 3 – MS/MS Data analysis
Spectrum from MALDI analysis 2
Open shareware for PMF 3 4
Online search with sequence databases
Best fit – Score histogram 5

9. Definitions of the components: Part 2 – Peptide Mass Fingerprinting (PMF)1
1. Peptide Mass Fingerprinting: This is one of the protein analysis methods which compares mass values of peptides generated from the protein analyte to a database of known proteins to arrive at its probable identity in the form of the “best fit”.
2. Spectrum from MALDI analysis: The peptide fragments generated after proteolytic digestion are analyzed by MALDI-TOF and the spectrum generated used for further analysis using online sequence databases.
3. Online search: Several open source databases are available online, which allow analysis of the MS spectrum generated.
4. Open shareware for PMF: These are database search algorithms used for comparing experimental masses against theoretically calculated peptide masses derived by applying “cleavage rules” to large primary sequence protein databases. The result of the comparison lists a number of proteins in the order of the best probable identity as derived by a probability score. The open shareware consists of the following fields which need to entered by the user:
Name and Used for identification of search entry and also for ing results page in case of loss of connection without requiring re-entry of data.
Search Title: Used to identify and label search entry and typically includes the name of the protein whose information is required.
Database/s: The primary sequence protein databases, including NCBInr and SwissProt against whom the query is run. A contaminants database is also recommended to eliminate contaminants such as keratin, trypsin and BSA. 2 3 4 5

10. Definitions of the components: Part 2 – Peptide Mass Fingerprinting (PMF)1
Taxonomy: It allows the search query to be limited to a particular species or a group of species bringing otherwise weaker hits to notice.
Enzyme: The proteolytic enzyme chosen during sample prep of analyte protein before its mass spectrometric analysis. Most popular of these is trypsin but if any other enzyme is used its site specificity is expected to be equal to or better than that of trypsin.
Missed Cleavage Allowed: Occurrence of partial digests during trypsinization of analyte protein at one or two Arginine and Lysine sites is a common phenomenon and needs to be accounted for during search against calculated peptide masses.
Modifications: During sample prep for Mass Spec Analysis of proteins, some changes in the mass of specific residues might occur, such as oxidation of methionine, carboxymethyl and cysteine etc. To account for these mass changes, the algorithm allows two types of modifications to be pre-selected- Fixed and Variable.
Fixed Modifications: Modifications that need to be applied collectively across the database to account for change in mass of specific residue/s. Most common fixed modification is the selection of the mass of carboxymethyl over cysteine replacing its mass as 161 Da.
Variable Modifications: These are mass changes suspected to occur during sample handling and accounted for by increasing the number of primary sequences compared against experimental masses. Most common variable modification is the oxidation of methionine residue in the analyte protein.
Protein Mass: Mass of intact protein in the form of a contiguous stretch including all matched peptides. If mass is unknown, this parameter can be left empty and the mass will remain unrestricted. 2 3 4 5

11. Definitions of the components: Part 2 – Peptide Mass Fingerprinting (PMF)1
Peptide Tolerance: This is a parameter associated with accuracy and resolution of the mass spectrometer and is used to account for shifts in isotope spacings.
Mass Values: To specify the type of charge of the analyte being examined by Peptide Mass Fingerprinting, i.e. MH+ , M-H- or if the masses correspond to neutral values like Mr .
Monoisotopic Mass Vs Average Mass Value: Depending upon the mass accuracy of a spectrometer, the experimental masses calculated for identification of analyte by Peptide mass fingerprinting is either chosen to be monoisotopic mass or the average mass of its isotopic elements. The selection of monoisotopic mass rests upon the ability of the instrument to resolve isotopes, and accurately determine peak mass. Average mass is the sum of abundance-weighted masses of all isotopes while the monoisotopic mass is the sum of masses of the most abundant isotope of each element. If the instrument has insufficient mass resolution capabilities combined with poor signal to noise ratio, the peptide mass of experimental values must be selected as being average to provide better identification.
5. Best fit – Score histogram: The “best fit” is defined as the primary identification of the analyte protein made by the database search algorithm representing either the exact protein being analyzed or the protein with the closest primary sequence homology, unusually with equivalent function in a related species. The score histogram depicts the distribution of protein scores for all the hits obtained by the query. 2 3 4 5

12. 3 Part 2, Step 1 1 2 4 5 Action Description of the action
Data input
Your name
Proteomics
Search title
Serum albumin
Database(s)
SwissProt
NCBInr
MSDB
Enzyme
Trypsin
Trypsin
Chymotrypsin
Peptidase 2
Taxonomy
Mammalian
Mammalian
Bacterial
Plant
Fixed modifications
Carbamoylation
Alkylation 3
Variable modification
Oxidation (M)
Protein mass 66
kDa
Peptide tol.
0.2 Da
Mass value
MH+ M
M-H-
Monoisotopic
Average
Data file
Choose file 4
Start search…
Action
Description of the action
Audio Narration
As shown in animaion.
First show the computer with the screen having a form on the inside. This must be zoomed into and the form above must be displayed. Each of the fields must be filled in as shown with some requiring selection using the white mouse pointer as depicted.
There are many MS analysis softwares available online that allow data generated from MS to be analyzed. They require inputs from the user regarding the experimental parameters used such as enzyme cleavage, protein name, fixed modifications etc. and the desired search criteria like taxonomy, peptide tolerance, taxonomy etc. Commonly used protein databases against which the MS information is processed to retrieve sequence data include NCBI, MSDB and SwissProt. The data file generated from MS is uploaded and the search carried out. We will demonstrate data analysis using Mascot ( 5
Source:

13. 3 Part 2, Step 2 1 2 4 5 Mascot Search Results Mascot Score Histogram
Data output
Mascot Search Results
User: Proteomics
Search title: Transcription factor
Database: SwissProt
Time stamp: 2 June 2010 at 17:45:35 GMT
Top score: 192 for PML_mouse, probable transcription factor
Mascot Score Histogram 2 3
>5% Random match
<5% Random match 4
Action
Description of the action
Audio Narration
As shown in animaion.
First show the computer with the screen displaying the search results. This must be zoomed into to clearly depict the report as shown. The arrows with the red text boxes must then appear.
The final results of the search are depicted in a concise report, beginning with a Protein Score Histogram. The protein score is a measure of the statistical significance of the protein hit. The histogram seen here displays the distribution of protein scores . Random matches made during database comparison are generally found in the green shaded region where the probability of finding a random hit is greater than 5%. The single red peak at the end of the histogram is the protein that has less than 5% chance of being a random hit, making it a statistically significant identity of the unknown protein analyte. 5

14. 3 Part 2, Step 3 1 2 4 5 Concise Protein Summary Report Action
Data output
Concise Protein Summary Report
PML_MOUSE Mass: Score: Expect: 1e-14 Matches: 15
Probable transcription factor PML for mouse
MURC_IDILO Mass: Score: Expect: 2 Matches: 5
UDP-N-acetylmuramate--L-alanine ligase (EC ) (UDP-N-acetylmuramoyl-L-alanine synthetase) - I
DPO1_RICHE Mass: Score: Expect: 2.8 Matches: 6
DNA polymerase I (EC ) (POL I) - Rickettsia helvetica
THIO_PONPY Mass: Score: Expect: 20 Matches: 3
Thioredoxin (Trx) - Pongo pygmaeus (Orangutan)
RBL2_RHOS4 Mass: Score: Expect: 28 Matches: 4
Ribulose bisphosphate carboxylase (EC ) (RuBisCO) - Rhodobacter sphaeroides (strain ATCC 17
RBL2_RHOSH Mass: Score: Expect: 28 Matches: 4
Ribulose bisphosphate carboxylase (EC ) (RuBisCO) - Rhodobacter sphaeroides (Rhodopseudomon
GPA1_YEAST Mass: Score: Expect: 29 Matches: 4
Guanine nucleotide-binding protein alpha-1 subunit (GP1-alpha) - Saccharomyces cerevisiae (Baker's
BNA4_YEAST Mass: Score: Expect: 36 Matches: 4
Kynurenine 3-monooxygenase (EC ) (Kynurenine 3-hydroxylase) (Biosynthesis of nicotinic aci
SWR1_DEBHA Mass: Score: Expect: 45 Matches: 6
Helicase SWR1 (EC ) - Debaryomyces hansenii (Yeast) (Torulaspora hansenii)
IFNW1_HUMAN Mass: Score: Expect: 69 Matches: 3
Interferon omega-1 precursor (Interferon alpha-II-1) - Homo sapiens (Human)        2 3
Protein information 4
Action
Description of the action
Audio Narration
As shown in animaion.
First show the computer with the screen with the search results displayed on the screen. This must be zoomed into to clearly depict it. The green box must then appear and flash along with the arrow and label. The user must be allowed to click on this and is taken to the next slide.
The Concise Summary report provides details of the peptide matches made by the algorithm which deduces the most probably protein match. The first hit is usually the “best fit” to the experimental masses that were entered in the search query. A protein score higher than 67 is considered to be a significant score. And a lower E value indicates that the probability of the hit being a random event is extremely low. Significant amount of information about the protein can be obtained from the report by clicking on the corresponding protein link. 5

15. 3 Part 2, Step 4 (a) 1 2 4 5 Action Description of the action
The protein score is a sum of the highest ion scores for each sequence, with duplicate matches being excluded. Score above 67 is significant for this hit.
Part 2, Step 4 (a) 1
Protein information – data analysis & interpretation
Predicted mass of the protein.
Match to: PML_MOUSE Score: 192 Expect: 1e-14
Probable transcription factor PML
Nominal mass (Mr): 97470; Calculated pI value: 5.88
NCBI BLAST search of PML_MOUSE against nr
Unformatted sequence string for pasting into other applications
Taxonomy: Mus musculus
Cleavage by Trypsin: cuts C-term side of KR unless next residue is P
Number of mass values searched: 18
Number of mass values matched: 15
Sequence Coverage: 22%
Matched peptides shown in Bold Red
1 MEPAPARSPR PQQDPARPQE PTMPPPETPS EGRQPSPSPS PTERAPASEE
51 EFQFLRCQQC QAEAKCPKLL PCLHTLCSGC LEASGMQCPI CQAPWPLGAD
101 TPALDNVFFE SLQRRLSVYR QIVDAQAVCT RCKESADFWC FECEQLLCAK
151 CFEAHQWFLK HEARPLAELR NQSVREFLDG TRKTNNIFCS NPNHRTPTLT
201 SIYCRGCSKP LCCSCALLDS SHSELKCDIS AEIQQRQEEL DAMTQALQEQ
251 DSAFGAVHAQ MHAAVGQLGR ARAETEELIR ERVRQVVAHV RAQERELLEA
301 VDARYQRDYE EMASRLGRLD AVLQRIRTGS ALVQRMKCYA SDQEVLDMHG
351 FLRQALCRLR QEEPQSLQAA VRTDGFDEFK VRLQDLSSCI TQGKDAAVSK
401 KASPEAASTP RDPIDVDLPE EAERVKAQVQ ALGLAEAQPM AVVQSVPGAH
451 PVPVYAFSIK GPSYGEDVSN TTTAQKRKCS QTQCPRKVIK MESEEGKEAR
501 LARSSPEQPR PSTSKAVSPP HLDGPPSPRS PVIGSEVFLP NSNHVASGAG
551 EAEERVVVIS SSEDSDAENS SSRELDDSSS ESSDLQLEGP STLRVLDENL
601 ADPQAEDRPL VFFDLKIDNE TQKISQLAAV NRESKFRVVI QPEAFFSIYS
651 KAVSLEVGLQ HFLSFLSSMR RPILACYKLW GPGLPNFFRA LEDINRLWEF
701 QEAISGFLAA LPLIRERVPG ASSFKLKNLA QTYLARNMSE RSAMAAVLAM
751 RDLCRLLEVS PGPQLAQHVY PFSSLQCFAS LQPLVQAAVL PRAEARLLAL
801 HNVSFMELLS AHRRDRQGGL KKYSRYLSLQ TTTLPPAQPA FNLQALGTYF
851 EGLLEGPALA RAEGVSTPLA GRGLAERASQ QS
Protein view
Predicted isoelectric point of the protein.
Indicates the % of matching peptides.
All peptides are displayed with matching peptides indicated in red. 2 3 4
Action
Description of the action
Audio Narration
As shown in animaion.
Show all the text output. Next show the green highlighted boxes one at a time with the corresponding dialogue box appearing for each of the highlighted regions. The results on the next slide must also be displayed along with this page.
On selecting a particular protein link, the protein view provides details regarding the protein score, molecular weight, isoelectric point, the sequence coverage of the protein etc. The greater the percentage sequence coverage, more are the number of matching peptides for that particular protein. All sequences are displayed with the matching sequences being indicated in red. 5

16. 3 Part 2, Step 4 (b) 1 2 4 5 Action Description of the action
Protein information – data analysis
Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence
R.SPRPQQDPARPQEPTMPPPETPSEGR.Q
R.QPSPSPSPTER.A
R.APASEEEFQFLR.C
K.HEARPLAELR.N
R.DYEEMASR.L
R.LDAVLQR.I
R.LRQEEPQSLQAAVR.T
R.QEEPQSLQAAVR.T
R.TDGFDEFK.V
K.MESEEGKEAR.L
R.SSPEQPRPSTSK.A
K.AVSPPHLDGPPSPR.S
R.SPVIGSEVFLPNSNHVASGAGEAEER.V
R.ELDDSSSESSDLQLEGPSTLR.V
R.VLDENLADPQAEDRPLVFFDLK.I
Protein view 2
Observed molecular weight.
Indicates beginning & end of each peptide.
Experimental molecular weight.
Calculated molecular weight.
Sequence of peptide fragment. 3 4
Action
Description of the action
Audio Narration
As shown in animaion.
Show all the text output. Next show the green highlighted boxes one at a time with the corresponding dialogue box appearing for each of the highlighted regions. The results on the next slide must also be displayed along with this page.
Sequence of each peptide fragment processed in the database is displayed along with information regarding its molecular weight, starting and ending amino acid number and the number of missed cleavages during tryptic cleavage. All these data provides a comprehensive understanding of the protein being analyzed. 5

17. 1 Master Layout (Part 3) 2 3 4 5 This animation consists of 3 parts:
Part 1 – Typical proteomics experiment
Part 2 – Peptide Mass Fingerprinting (PMF)
Part 3 – MS/MS Data analysis
Spectra from MS/MS analysis 2
Open shareware for MS/MS analysis 3 4
Online search with sequence databases
Peptide summary report 5

18. Definitions of the components: Part 3 – MS/MS data analysis1
1. Tandem MS/MS analysis: This is another protein analysis method which compares the fragmentation spectra of the analyte protein. These fragmentation and parent masses, representative of the amino acid sequence of the analyte’s peptides are then compared to databases of known proteins to identify each peptide at a time and then infer protein identity by searching for the presence of particular peptides.
2. Spectrum from MS/MS analysis: MS/MS analysis generates fragmentation patterns for each peptide of the proteolytic digest. These are useful for determining the sequence of the protein analyte.
3. Online search: Several open source databases are available online, which allow analysis of the MS spectrum generated.
4. Open shareware for MS/MS analysis: This consists of a two step process involving; first, the identification of peptides by comparing sequenced peptides against theoretical databases of MS/MS Spectra generated from primary sequence databases and second, by collating these peptide identifications into a minimal protein list and scoring them to provide statistical validation. In addition to the same fields discussed for PMF, this shareware consists of the following additional fields which need to entered by the user:
Database/s: The databases available for MS/MS spectra comparison, include NCBInr Db, SwissProt Db apart from several EST databases if the initial search provides no positive Ids. Selecting a contaminants database is also recommended to eliminate contaminants such as keratin, trypsin and BSA. 2 3 4 5

19. Definitions of the components: Part 3 – MS/MS data analysis1
Quantitation: It is a search parameter used to implement different search protocols which might have been used to quantify protein analyte by mass spectrometry. Some examples of the options available for setting a particular quantitation method include, iTRAQ 4plex, SILAC multiplex, ICAT D8 etc.
Precursor Value: This parameter calls for the m/z value of the parent peptide in case the MS/MS data format does not automatically provide it. It is used, in conjunction with the charge of the parent peptide, to calculate its relative molecular weight (Mr).
Peptide Charge: It is the parameter used to indicate the charge state of the precursor peptide, so that its Mr can be calculated from the observed m/z value.
MS/MS Tolerance: It is associated with accuracy and resolution of the mass spectrometer and used to resolve isotope shifts in MS/MS fragmentation masses.
Instrument: Informing the algorithm about the instrument used to carry out fragmentation studies helps especially when instead of just CID, either ETD or ECD has been used. Depending upon the instrument a particular ion stream is used to find a peptide match.
Data Format: There are several data formats that are used to process MS/MS fragmentation data such as SCIEX API III, PerSeptive (.PKS) and Bruker (.XML) associated with software or instrument. Depending upon the search type, individual MS/MS spectrum or thousands of spectra from LC-MS/MS type search can be carried out. 2 3 4 5

20. Definitions of the components: Part 3 – MS/MS data analysis1
Error Tolerant Search: This parameter can be put to use in case, a large percent of the experimental MS/MS remains unidentified. By performing this type of search, it is possible to make adjustments to accommodate issues such as absence of peptide sequence in database, non-specificity of proteolytic enzyme used for protein digestion or even unknown post-translational modifications that cause fluctuations in the mass of analyte isomers.
5. Peptide summary report: The peptide summary report provides the most probable protein identity by individually identifying and grouping each of the peptides. The greater the number of peptides, the higher the protein score for the hit as it is derived from individual ion scores. Further statistical validations will help ascertain the find and improve the statistical health of the protein hit. 2 3 4 5

21. 3 Part 3, Step 1 1 2 4 5 Action Description of the action
Data input
Your name
Proteomics
Search title
Sample protein
Database(s)
SwissProt
NCBInr
MSDB
Enzyme
Trypsin
Trypsin
Chymotrypsin
Peptidase
Quantitation 2
Taxonomy
Bacterial
Mammalia
Bacterial
Plant
Fixed modifications
Carboxymethyl (C) 3
Variable modification
Oxidation (M)
Peptide tol.
1.2 Da
# C13
MS/MS tol.
0.2 Da
Peptide charge
Monoisotopic
Average
Data file
Choose file
Data format
Precursor 4
Instrument
ESI-Q-TOF
Start search…
MALDI-TOF
ESI-Q-TOF
MALDI-TOF-TOF
Action
Description of the action
Audio Narration
The MS/MS data analysis shareware has some extra inputs such as Quantitation, MS/MS tolerance, peptide charge, instrument etc. in addition to the fields for PMF. They require inputs from the user regarding the experimental parameters used such as enzyme cleavage, protein name, modifications etc. and the desired search criteria like taxonomy, peptide tolerance etc. Commonly used protein databases against which the MS information is processed to retrieve sequence data include NCBI, MSDB and SwissProt. The data file generated from MS is uploaded and the search carried out.
As shown in animaion.
First show the computer with the screen having a form on the inside. This must be zoomed into and the form above must be displayed. Each of the fields must be filled in as shown with some requiring selection using the white mouse pointer as depicted. 5

22. 3 Part 3, Step 2 1 2 4 5 Mascot Search Results Mascot Score Histogram
Data output
Mascot Search Results
User: proteomics
Search title: Sample protein
Database: NCBInr
Taxonomy: Mammalia
Time stamp: 2 June 2010 at 17:45:35 GMT
Protein hits:
Mascot Score Histogram 2 3
>5% Random match
<5% Random match 4
Action
Description of the action
Audio Narration
As shown in animaion.
First show the computer with the screen displaying the search results. This must be zoomed into to clearly depict the report as shown. The red box must appear at the region indicated along with the blue arrow.
The Tandem MS protein analysis is used to obtain protein identities from each of the sequenced peptides. The results page begins with a list of probable protein identities and their respective sources. The score histogram provides details similar to the PMF analysis, with the probability distribution being displayed graphically. The green shaded region is indicative of a match that has greater than 5% chance of being random while the red peak indicates that the chances of a random match is less than 5%. 5

23. 3 Part 3, Step 3 1 2 4 5 Peptide summary report
Data output
Peptide summary report
gi| Mass: Score: Matches: 8(3) Sequences: 3(2)
Unknown (protein for IMAGE: ) [Homo sapiens]
Check to include this hit in error tolerant search
Query Observed Mr(expt) Mr(calc) ppm Miss Score Expect Rank Unique Peptide
U K.FGEAVWFK.A
(40) U K.FGEAVWFK.A
(32) U K.FGEAVWFK.A
e U R.WAMLGALGCVFPELLAR.N + Oxidation (M)
(48) U R.WAMLGALGCVFPELLAR.N + Oxidation (M)
U R.LAMFSMFGFFVQAIVTGK.G + Oxidation (M)
(35) U R.LAMFSMFGFFVQAIVTGK.G + Oxidation (M)
(22) U R.LAMFSMFGFFVQAIVTGK.G + 2 Oxidation (M)
gi| Mass: Score: Matches: 3(0) Sequences: 2(0)
zona pellucida sperm-binding protein 4 [Sus scrofa]
Query Observed Mr(expt) Mr(calc) ppm Miss Score Expect Rank Unique Peptide
U K.GPGSSMGVEASYR.G
(21) U K.GPGSSMGVEASYR.G
e U K.YSRPPVDSHALWVAGLLGSLIIGALLVSYLVFRK.W 2 3
Protein information
Peptide information 4
Description of the action
Action
Audio Narration
As shown in animaion.
First show the computer with the screen displaying the search results. This must be zoomed into to clearly depict the report as shown. The green highlight boxes must then appear with their labels. User must be allowed to click on these highlighted regions. Clicking on ‘protein information’ must redirect user to steps 4 (a) & (b) while ‘peptide information’ must redirect user to steps 5(a) & (b).
The summary report lists all the protein matches obtained from the database search with their respective molecular weight, protein score, source organism and details regarding each of its fragmented peptides. Further information about any of the protein sequences can be obtained by clicking on the corresponding protein link. Data regarding each of the peptide fragmentation patterns can also be obtained by clicking on the peptide link indicated by the query number. 5

24. 3 Part 3, Step 4 (a) 1 2 4 5 Mascot search results Action
The protein score is a sum of the highest ion scores for each sequence, with duplicate matches being excluded. A score above 67 is considered significant. In this case. 1
Protein information – data analysis & interpretation
Match to: gi| Score: 225
Unknown (protein for IMAGE: ) [Homo sapiens]
Found in search of C:\Users\harini\Desktop\MS\3C.LC-MS-MS data analysis Raw data file- mgf files\Data file1.mgf
Nominal mass (Mr): 30840; Calculated pI value: 6.00
NCBI BLAST search of gi| against nr
Unformatted sequence string for pasting into other applications
Taxonomy: Homo sapiens
Links to retrieve other entries containing this sequence from NCBI Entrez:
gi| from Homo sapiens
Fixed modifications: Carbamidomethyl (C)
Variable modifications: Oxidation (M)
Cleavage by Trypsin: cuts C-term side of KR unless next residue is P
Sequence Coverage: 14%
Matched peptides shown in Bold Red
1 HHHSPTLREH GRRTRTSLLE AMATTAMALS PSSFAGKAVK DLPSSALFGE
51 ARVTMRKTAA KAKPVSSGSP WYGSDRVLYL GPLSGDPPSY LTGEFPGDYG
101 WDTAGLSADP ETFAKNRELE VIHCRWAMLG ALGCVFPELL ARNGVKFGEA
151 VWFKAGSQIF SEGGLDYLGN PSLVHAQSIL AIWACQVVLM GAVEGYRVAG
201 GPLGEIVDPL YPGGSFDPLG LADDPEAFAE LKVKEIKNGR LAMFSMFGFF
251 VQAIVTGKGP LENLADHLSD PVNNNAWAFA TNFVPGK
Mascot search results
Protein view
Predicted mass of the protein.
Predicted isoelectric point of the protein. 2
Indicates the % of matching peptides.
All peptides are displayed with matching peptides indicated in red. 3 4
Action
Description of the action
Audio Narration
As shown in animaion.
Show all the text output. Next show the green highlighted boxes one at a time with the corresponding dialogue box appearing for each of the highlighted regions. The results on the next slide must also be displayed along with this page.
The protein view obtained on selecting a particular protein link, is very similar to the protein view observed in PMF. It provides details regarding the protein score, molecular weight, isoelectric point, the sequence coverage of the protein etc. Protein scores above 67 are considered significant and greater the percentage sequence coverage, more are the number of matching peptides for that particular protein. All sequences are displayed with the matching sequences being indicated in red. 5

25. 3 Part 3, Step 4 (b) 1 2 4 5 Mascot search results Action
Protein information – data analysis & interpretation
Start - End Observed Mr(expt) Mr(calc) ppm Miss Sequence
R.WAMLGALGCVFPELLAR.N Oxidation (M) (Ions score 118)
R.WAMLGALGCVFPELLAR.N Oxidation (M) (Ions score 48)
K.FGEAVWFK.A (Ions score 66)
K.FGEAVWFK.A (Ions score 40)
K.FGEAVWFK.A (Ions score 32)
R.LAMFSMFGFFVQAIVTGK.G Oxidation (M) (Ions score 42)
R.LAMFSMFGFFVQAIVTGK.G Oxidation (M) (Ions score 35)
R.LAMFSMFGFFVQAIVTGK.G 2 Oxidation (M) (Ions score 22)
Mascot search results
Protein view
Indicates score of each ion fragment. Used for calculation of the protein score. 2
Indicates beginning & end of each peptide.
Observed molecular weight.
Experimental molecular weight.
Calculated molecular weight.
Sequence of peptide fragment. 3 4
Action
Description of the action
Audio Narration
As shown in animaion.
Show all the text output. Next show the green highlighted boxes one at a time with the corresponding dialogue box appearing for each of the highlighted regions.
Information about each of the matched peptides is also displayed. The start and end amino acid positions, calculated and experimental molecular weights, number of missed tryptic cleavages, sequence of each peptide fragment and their corresponding ion scores are shown. The highest ion scores are used for computing the final protein score. 5

26. 3 Part 3, Step 5 (a) 1 2 4 5 Mascot search results Action
Peptide sequence whose fragmentation pattern is shown.
Peptide information – data analysis and interpretation
Mascot search results
Peptide view
MS/MS Fragmentation of FGEAVWFK
Found in gi| , Unknown (protein for IMAGE: ) [Homo sapiens]
Match to Query 4: from( ,2+) intensity( )
Title: Sum of 11 scans in range 1333 (rt= , f=2, i=174) to 1373 (rt= , f=2, i=184) [\\Qtof\Qtof 17\JAN2004.PRO\Data\6p013-sanjeeva-10.raw]
Data file C:\Users\harini\Desktop\MS\3C.LC-MS-MS data analysis Raw data file- mgf files\Data file1.mgf
Range values for the x-axis that can be modified by the user to zoom in or zoom out of the graphical representation. 2 3 4
Action
Description of the action
Audio Narration
As shown in animaion.
Show all the text output. Next show the green highlighted boxes one at a time with the corresponding dialogue box appearing for each of the highlighted regions.
Each peptide in Tandem MS/MS undergoes a second round of fragmentation when it passes through the second mass analyzer before it reaches the detector. This provides significantly larger amount of information regarding each peptide fragment. This can be viewed by clicking on the peptide links provided in the summary report. The fragmentation pattern is displayed graphically, which can be zoomed into as per the requirement by adjusting the x-axis plot values. 5

27. 3 Part 3, Step 5 (b) 1 2 4 5 Mascot search results Action
Peptide information – data analysis & interpretation
Mass of the peptide fragment displayed.
Mascot search results
Peptide view
Monoisotopic mass of neutral peptide Mr(calc):
Fixed modifications: Carbamidomethyl (C) (apply to specified residues or termini only)
Ions Score: 66 Expect:
Matches : 23/78 fragment ions using 16 most intense peaks (help)
Amino acid sequence obtained through computation using y-ion and b-ion values.
b-ions: Ions formed with charge retained on N-terminal.
y-ions: Ions formed with positive charge retained on C-terminal.
b1 ( ) – b2 ( ) =  G 2
y7 ( ) – y6 ( ))=  G #
Immon a a0 b b0
Seq y y* y0 1 F 8 2 G 7 3 E 6 4 A 5 V W K 3
b6 ( ) – b7 ( ) =  F
y2 ( ) - y1 ( ) =  F 4
Action
Description of the action
Audio Narration
As shown in animaion.
Show all the text output. Next show the green highlighted boxes one at a time with the corresponding dialogue box appearing for each of the highlighted regions.
At low collision energy, each peptide fragment is cleaved at the amide bond which can result in the formation of two types of ions – the y ion & b ion. In y-ions, the positive charge is retained on the C-terminus of the peptide ion while in b-ions, charge is retained on the N-terminal. These ion masses can be used to compute the amino acid sequence by calculating the mass difference between consecutive ions. Each mass difference value corresponds to a particular amino acid, which can be obtained from a standard information table. The y-ion series & the b-ion series run opposite to each other as indicated in the example above. 5

28. Interactivity option 1:Step No:1
Based on the mass values indicated in the graph shown below and the table provided showing the average and monoisotopic mass of each amino acid, deduce the sequence of this peptide fragment.
242
402
473
601
530
m/z 25 50 75
100
Relative Abundance 72
171
299
769 2 3 4
Interacativity Type
Options
Boundary/limits
Results
The correct answer is D. If user chooses this, it must turn green with the message ‘right answer’. If he chooses any of the others, it must turn red, with the message ‘wrong answer’.
The graph above with all values & the table shown in the next slide must be displayed. The four option must be shown & user must be allowed to choose any 1 of the 4 options. 5
Choose the correct answer.

29. Interactivity option 2:Step No:21
Amino acid LC SLC Average Monoisotopic
Glycine Gly G
Alanine Ala A
Serine Ser S
Proline Pro P
Valine Val V
Threonine Thr T
Cysteine Cys C
Leucine Leu L
Isoleucine Ile I
Asparagine Asn N
Aspartic acid Asp D
Glutamine Gln Q
Lysine Lys K
Glutamic acid Glu E
Methionine Met M
Histidine His H
Phenyalanine Phe F
Arginine Arg R
Tyrosine Tyr Y
Tryptophan Trp W 2 3 4 5
Answers:
A) AVAGCGGAF
C) AVACCAGAY
B) STAGTAGAR
D) AVAGCAGAR

30. Questionnaire 1
1. Which one of these is common across all Mass Spec based proteomics experiments carried out? A) Liquid Chromatography B) Proteolysis C) 2-D Gel Electrophoresis D) Isoelectric Focusing 2. Peptide Mass Fingerprinting or PMF is defined as? A) Finding the best fit for peptides identified by fragmentation. B) Finding the best fir for protein by sequencing in a Triple Quadrupole Analyzer. C) Finding fingerprints of proteins on 2-DE Gels. D) Finding the best fit for masses of peptides identified by MALDI-TOF. 3. Which one of these mass values represents a protein/peptide ion? A) M-H- B) M-H+ C) MH+ D) MH- 4. The average mass of which of the following amino acids corresponds to ? A) Serine B) Glycine C) Alanine D) Glutamine 2 3 4 5

31. Links for further reading
Reference websites:
– The most popular Open shareware site for processing PMF and Tandem Mass Spectrometric data called MASCOT is available here.
Research papers:
1. Henzel.W.J., Watanabe.C., Stults.J.T. (2003). Protein Identification: The Origins of Peptide Mass fingerprinting. J Am Soc Mass Spectrom., 14(9)., pp:
2. Nesvizhskii , A.I., Vitek, O., Aebersold, R. (2007). Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat.Methods., 4(!0), pp
3. Deutsch, E.W., Lam, H., Abersold, R. (2008) Data analysis and bioinformatics tools for tandem mass spectrometry in proteomics. Physiol Genomics. 33 (1), pp:18-25.
4. Yates, JR., Mass Spectrometry and the Age of Proteome. J.Mass.Spec., 33(1), pp.1-19.