# Presentation: 517947

## Mass Spectrometry: Characterizing Proteins, Complexes, and Cells 

**Dr. Timothy D. Veenstra**
**Director, Laboratory of Proteomics and Analytical Technologies and**
**NCI-Frederick Biomedical Proteomics Program**

## The Global Revolution

**Genomics         ********     Transcriptomics    ********        Proteomics**

**The Global Revolution**

## Proteomics

**Proteomics**

## Mass Spectrometry-based Proteomics

**High Mass Accuracy (~ 1-10 ppm)**
**High Resolution (~25,000 – 500,000)**
**High Sensitivity (routinely low femtomole)**
**Tandem Mass Spectrometry for Peptide Identification**

**Mass Spectrometry-based Proteomics**

## From Lysates to Identified Proteins

**From Lysates to Identified Proteins**

## A)  Protein Identification

**B)  Identification of Post-translational Modifications**
**  ****Protein Quantitation**
**  ****Biomarker Identification**

**Major Foci of Mass Spectrometry-based Proteomics**

## Analyte Analysis Using Mass Spectrometry

- Atmospheric Pressure Chemical Ionization
- Electron Ionization

**Analyte Analysis Using Mass Spectrometry**

- Gas Chromatography
- Liquid Chromatography

- Quadrupole
- Ion Trap
- Time-of-Flight

## Ionization Sources

- Sample solution

- electrospray capillary

- high voltage applied

- Atmospheric Pressure

- High Vacuum

**A. Electrospray Ionization (ESI)**

**B. Matrix assisted laser/desorption ionization (MALDI)**

- orifice

- sample and matrix

- pulsed laser light

- peptide and  matrix ions
- desorbed from matrix

- MALDI target
- plate

- orifice

- Quadrupole

**Ionization Sources**

## Mass Analyzer

***Purpose: to separate and detect sample ions***
- The main function of the **mass analyzer** is to **separate**, or **resolve**, ions formed in the ionization source of the mass spectrometer according to their **mass-to-charge (m/z)** ratios.
-  There are a number of mass analyzers available:
- ion-trap
- Linear ion-trap
- Time-of-flight (TOF)
- Triple-quadrupole
- Fourier transform ion cyclotron resonance (FTICR)

## Characteristics of Different Types of Mass Analyzers

| Analyzer | Sensitivity | Resolution | Mass accuracy |
| --- | --- | --- | --- |
| Ion-trap | Good | Low | Low |
| Linear ion-trap | Excellent | Low | Low |
| Triple-quadrupole | Good | Good | Good |
| TOF | Good | High | High |
| FTICR | Excellent | High | High |

**Characteristics of Different Types of Mass Analyzers**

## Liquid Chromatography-Mass Spectrometry (LC-MS)

- 10 cm reversed-phase LC electrospray column
- (50 m ID/3m particles)

**h******

**IT-MS**

**Liquid Chromatography-Mass Spectrometry (LC-MS)**

## Microcapillary Liquid Chromatography Column

**Microcapillary Liquid Chromatography Column**

## Slide 13

## Slide 14

## Mass Spectrometry Characterization of Protein Complexes

## Experimental Design 

**KSR1**

**Marker**

**Control**

**KSR1**

**Experimental Design **

**IP KSR1**
**Examine **
**complexes **
**by SDS-PAGE**
**Mass Spectrometry**

- Immuno-precipitation

- Pyo antibody

## Identification of Proteins Complexes

**Marker**

**Control**

**KSR**

- In-gel digestion

- Extract peptides

- MALDI-TOF/TOF
- analysis

**Identification of Proteins Complexes**

- LC-tandem MS

## *

**800**

**1340**

**1880**

**2420**

**2960**

**3500**

**Mass (*****m/z*****)**

**10**

**20**

**30**

**40**

**50**

**60**

**70**

**80**

**90**

**100**

**% Intensity**

- 1256.6006

- 1189.6686

- 1819.9570

- 1447.7240

- 1205.6603

- 1524.7152

- 2087.9832

- 842.5067

- 1143.2697

- 917.5259

- 1360.6909

- 1773.6384

- 1086.6537

- 1278.5853

- 1678.9277

- 2180.9668

- 2331.2314

- 1575.8091

- 2808.3474

- 2866.4988

**14-3-3 epsilon**

**Protein Identification by Peptide Mapping**

- Experimental
- Mass spectrum

- Theoretical
- mass spectrum

- Tryptic peptides

- Theoretical
- tryptic peptides

- Protein

- In silico protein
- sequence

- ECDELIPGKTYFLLRAVLEDGKCCFGLWAVTKVMTYHILRYEDSQTKEFGILPDRESS

- ECDELIPGK
- TYFLLR
- AVLEDGK
- CCFGLWAVTK
- VMTYHILR
- YEDSQTK
- EFGILPDR
- ESS

- ECDELIPGK
- TYFLLR
- AVLEDGK
- CCFGLWAVTK
- VMTYHILR
- YEDSQTK
- EFGILPDR
- ESS

- ECDELIPGKTYFLLRAVLEDGKCCFGLWAVTKVMTYHILRYEDSQTKEFGILPDRESS

## L E E L E L D E Q Q R

- ESI

- MS-1

- MS-2

- Collision
- cell

- Detector

**L E E L E L D E Q Q R**

- Collision cell

**L E E L E L D E Q Q **

**L E E L E L D E Q**

**L E E L E L D E**

**L E E L E L D**

**L E E L E L**

**L E E L E**

**L E E L**

**L E E**

**E E L E L D E Q Q **

**E L E L D E Q Q **

**L E L D E Q Q **

**E L D E Q Q **

**L D E Q Q **

**D E Q Q **

**E Q Q **

**Q Q **

**Protein Identification by Tandem Mass Spectrometry (MS/MS)**

## Fragmentation along the amide bonds creates amino acid “ladders” that can be used to re-create the sequence of the peptide.

- 200

- 300

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

- 1300

- 1400

- 1500

***m/z***

- 10

- 20

- 30

- 40

- 50

- 60

- 70

- 80

- 90

- 100

- Relative Abundance

- E

- E

- E

- L

- L

- L

- D

- E

- QQR

- y10

- y9

- y8

- y7

- y6

- y5

- y4

- y3

- y11

**Fragmentation along the amide bonds creates amino acid “ladders” that can be used to re-create the sequence of the peptide.**

## + another ~50 proteins identified by a single peptide

**Accession**

**Protein**

**Peptide**

**MH+**

**Charge**

**XC**

- P07225

- Vitamin K-dependent protein S precursor

- K.IETISHEDLQR.Q

- 1340.6805

- 2

- 3.6267

- K.DVDECSLKPSICGTAVCK.N

- 1867.8598

- 2

- 3.1124

- K.EAVMDINK.P

- 919.4553

- 1

- 2.1308

- K.EAVMDINKPGPLFKPENGLLETK.V

- 2540.3429

- 3

- 5.2995

- K.HCLVTVEK.G

- 928.4921

- 2

- 3.2371

- K.NGFVMLSNK.K

- 1009.5135

- 1

- 2.6444

- K.PENGLLETK.V

- 1000.531

- 1

- 3.0494

- K.PGPLFKPENGLLETK.V

- 1639.9054

- 2

- 3.431

- K.SQDILLSVENTVIYR.I

- 1749.9381

- 2

- 5.3785

- R.KVESELIKPINPR.L

- 1522.8952

- 2

- 4.5497

- K.VYFAGFPR.K

- 956.4989

- 2

- 2.4449

- R.QLAVLDK.A

- 786.472

- 1

- 2.1313

- K.VESELIKPINPR.L

- 1394.8002

- 2

- 3.7376

- R.FSAEFDFR.T

- 1018.4629

- 2

- 2.718

- R.SFQTGLFTAAR.Q

- 1198.6215

- 2

- 3.7887

- R.NNLELSTPLK.I

- 1128.6259

- 2

- 3.202

- R.QSTNAYPDLR.S

- 1164.5644

- 2

- 2.7292

- K.FRLPEISR.F

- 1017.584

- 2

- 2.7825

- P10515

- Dihydrolipoyllysine-residue acetyltransferase

- R.VVDGAVGAQWLAEFR.K

- 1617.8384

- 2

- 4.3297

- K.ISVNDFIIK.A

- 1048.6037

- 2

- 2.6772

- K.GVETIANDVVSLATK.A

- 1516.8217

- 2

- 4.5478

- K.DIDSFVPSK.V

- 1007.5044

- 1

- 2.1092

- K.VPEANSSWMDTVIR.Q

- 1604.7737

- 2

- 3.6597

- K.VPLPSLSPTMQAGTIAR.W

- 1738.952

- 2

- 4.5614

- P11021

- 78 kDa glucose-regulated protein precursor (GRP 78)

- K.SQIFSTASDNQPTVTIK.V

- 1836.9338

- 2

- 5.0211

- R.IINEPTAAAIAYGLDK.R

- 1659.8952

- 2

- 5.1962

- R.ITPSYVAFTPEGER.L

- 1566.7798

- 2

- 2.876

- P11142

- Heat shock cognate 71 kDa protein (Heat shock 70 kDa protein 8)

- K.SQIHDIVLVGGSTR.I

- 1481.8071

- 2

- 4.5857

- K.SINPDEAVAYGAAVQAAILSGDK.S

- 2260.1456

- 2

- 4.8969

- K.NQVAMNPTNTVFDAK.R

- 1649.7952

- 2

- 3.5025

- P38646

- Stress-70 protein, mitochondrial precursor (75 kDa glucose regulated protein) (GRP 75)

- R.QAASSLQQASLK.L

- 1231.6641

- 2

- 3.5859

- R.VINEPTAAALAYGLDK.S

- 1645.8796

- 2

- 5.3113

- K.NAVITVPAYFNDSQR.Q

- 1694.8497

- 2

- 2.6729

- K.MKETAENYLGHTAK.N

- 1592.7737

- 3

- 2.9914

- K.SDIGEVILVGGMTR.M

- 1446.7621

- 2

- 2.8238

- K.VQQTVQDLFGR.A

- 1290.6801

- 2

- 3.6916

- R.TTPSVVAFTADGER.L

- 1450.7172

- 2

- 3.6529

- R.QAVTNPNNTFYATK.R

- 1568.7703

- 2

- 3.1202

- K.SQVFSTAADGQTQVEIK.V

- 1808.9025

- 2

- 5.4229

- R.EQQIVIQSSGGLSK.D

- 1473.7907

- 2

- 3.8745

- R.ASNGDAWVEAHGK.L

- 1341.6182

- 2

- 2.8473

- R.AQFEGIVTDLIR.R

- 1361.7423

- 2

- 4.4901

- R.VEAVNMAEGIIHDTETK.M

- 1856.9059

- 2

- 4.6726

- K.LLGQFTLIGIPPAPR.G

- 1592.9523

- 2

- 3.3736

- K.ETAENYLGHTAK.N

- 1333.6383

- 2

- 3.0233

- K.DAGQISGLNVLR.V

- 1242.6801

- 2

- 4.0011

- K.STNGDTFLGGEDFDQALLR.H

- 2055.9618

- 2

- 4.5971

- Q8TB01

- Similar to cytoskeleton-associated protein 4

- K.IETNENNLESAK.G

- 1361.6543

- 2

- 3.2985

- R.LEGLGSSEADQDGLASTVR.S

- 1904.9196

- 2

- 2.9276

**+ another ~50 proteins identified by a single peptide**

## Identification of Phosphopeptides by MS/MS

- [M-H3PO4+2H]2+

- 0

- 100

- 200

- 300

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

- 1300

- 1400

***m/z***

- 10

- 20

- 30

- 40

- 50

- 60

- 70

- 80

- 90

- 100

**Relative Abundance (%)**

- b13

- b12

- b11

- b10

- y9

- b8

- b6

- y6

- y5

- y4

- b3

- y3

- y2

- b2

- y7

- y8

- y10

- y11

- y12

- Δ*m/z*=166.9

- pSer

**y10**

**y11**

- 0

- 100

- 200

- 300

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

- 1300

***m/z***

- 0

- 10

- 20

- 30

- 40

- 50

- 60

- 70

- 80

- 90

- 100

**Relative Abundance (%)**

- K.PNQSAFTSSGLVSK.M

- b2

- b3

- y2

- b4

- y4

- b5

- b6

- b8

- b9

- b10

- b11

- b12

- b13

- y6

- y7

- y8

- y9

- y10

- y11

- y12

- y13

**y10**

**y11**

- Δ*m/z*=87.1

- Ser

**Identification of Phosphopeptides by MS/MS**

## *

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

***m/z***

- 0

- 20

- 40

- 60

- 80

- 100

**Relative Abundance**

**923.8**

- MS

- 300

- 500

- 700

- 900

- 1100

- 1300

- 1500

- 1700

***m/z***

- 0

- 20

- 40

- 60

- 80

- 100

**Relative Abundance**

**874.8**

**[M-H****3****PO****4****+2H]****2+**** **

- MS/MS
- 923.8

**Spectrum indicates peptide is phosphorylated, however, the tandem MS data is insufficient to obtain the peptide’s sequence**

- 0

- 200

- 400

- 600

- 800

- 1000

- 1200

- 1400

- 1600

***m/z***

- 0

- 20

- 40

- 60

- 80

- 100

**Relative Abundance**

- b14

- b13

- y12

- b12

- b11

- y10

- b10

- y9

- b9

- y8

- y7

- b7

- b6

- y5

- y4

- b4

- b3

- y2

- b5

- b8

- y3

- y6

- y11

- y12++

- b15++

- K.T#LDTQLEIVIEILHK.E

- MS/MS/MS
- 874.8

*****

*****

**The Use of MS/MS/MS to Identify Phosphopeptides**

## Validation of MS Results is Absolutely Necessary

**Validation of MS Results is Absolutely Necessary**

**When identifying protein complexes by MS, many proteins that are not specific to the complex will also be identified.**
**Unfortunately the MS results do not give information related to the specificity of the protein.**
**MS essentially narrows down the possibilities concerning the proteins that may be part of the complex of interest.**
**Validation to ascertain biological significance is critical.**

## 2-D PAGE-BASED PROTEOMICS

**2-D PAGE**

## Solution-based proteomics 

**Data analysis and display**

## Fractionation of complex protein mixtures

**Subcellular fractionation:**
**Plasma membrane**
**Lipid rafts**
**Mitochondria**

## Multidimensional Separation/Tandem Mass Spectrometry Analysis

**Multidimensional Separation/Tandem Mass Spectrometry Analysis**

## Quantitative Proteomics Using Stable Isotope Labeling

**In global quantitative proteomic studies, the aim is to measure the relative proteins abundance between two distinct systems.**
**Stable isotope labeling is often used to enable the quantitative measurement of two proteins from the two systems.**
**The net result of such an analysis is a list of proteins and their relative abundance.  The measurement is somewhat analogous to mRNA array analysis, however, the process takes much more time (i.e. 3-4 weeks).**
**Unfortunately, the technology is unable to specifically focus on proteins that may be of interest (i.e. you get what you get).  **

## Quantitative Proteomic Schemes

**Quantitative Proteomic Schemes**

**Cell Culture**

**Cell Lysis**

**Stable Isotope **
**Labeling**

## Low Throughput

*m/z*

- NCSETQYESK

- ICAT

- MS Quantitation

**  ****9.03 Da**

*m/z*

- % Intensity

**Low Throughput**
**Permits an A vs. B comparison**

**Stable-Isotope Labeling**

**Quantitative Proteomics. I. Stable Isotope Labeling**

## Low throughput

**Allows an unlimited number **
**of comparisons**

**Quantitative Proteomics. II. Subtractive Proteomics**

## Very High Throughput

**Very High Throughput**
**Multi-sample Comparisons**
**Limited in Dynamic Range and Sensitivity**

**Quantitative Proteomics. III. Proteomic Patterns**

## +

**-****S-CH****2****-Protein**

**+**

_**Iodoacetamide Group**_
** ****reactive towards thiols**
** ****enables the modification of cysteinyl residues**

**The Versatile ICAT Reagent**

## ICAT Methodology

- (ICAT-13C0)

- Treated
- (ICAT-13C9)

- Pool
- Proteins

- MS/MS Identification

- Digest

- Enrich ICAT-peptides

- MS Quantitation

- Cleave biotin tags

- 400

- 800

- 1200

- 1600

*m/z*

**heavy**

**light**

**  ****9.03 Da**

- 2046.0

- 2050.6

- 2055.2

- 2059.8

- 2064.4

- 2069.0

*m/z*

- 25

- 50

- 75

- 100

- % Intensity

**ICAT Methodology**

## Measurement of Protein Relative Abundance

**Measurement of Protein Relative Abundance**

## ICAT Label

**Control MC3T3-E1**
**cells**

**Inorganic Phosphate-treated (10 mM) **
**MC3T3-E1 cells**

**ICAT Label**

**All cysteines labeled with ****13****C****0**** ICAT**

**All cysteines labeled with ****13****C****9**** ICAT**

**Combine and Proteolyze**

**Affinity Isolate ICAT Peptides**

**Strong Cation Exchange Fractionate ICAT Labeled Peptides**

**Analyze ICAT Peptide Fractions by ********RPLC-MS/MS**

**Quantitate by Integrating ICAT Peptide Elution Peak Areas**

**Identify proteins from sequence information from MS/MS**

- b7

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

- 1300

*m/z*

- Relative Abundance

- b3

- b4

- b6

- b8

- y3

- y4

- y5

- y6

- y7

- y8

**SHCIAEVEK**

**ICAT**

- Relative Abundance

- Retention time

**(6 x 10****6**** cells)**

## Multidimensional Separation/Tandem Mass Spectrometry Analysis

**Multidimensional Separation/Tandem Mass Spectrometry Analysis**

## Identification of ICAT-labeled Peptides

- 300

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

- 1300

- 1400

- 1500

- 1600

- 1700

- 1800

- 1900

***m/z***

- 0

- 50

- 100

**Relative Abundance**

- 704.3

- 1017.2

- 888.2

- 1387.5

- 591.3

- 1258.6

- 1130.4

- 1515.7

- 817.4

- 462.2

- 375.2

- 1017.5

- 300

- 400

- 500

- 600

- 700

- 800

- 900

- 1000

- 1100

- 1200

- 1300

- 1400

- 1500

- 1600

- 1700

- 1800

- 1900

***m/z***

- 0

- 50

- 100

- 704.3

- 591.3

- 817.5

- 888.5

- 1130.5

- 1258.5

- 1515.6

- 462.3

- 1387.5

- 288.2

**Relative Abundance**

- 375.2

- y13+

- y12+

- y11+

- y10+

- y6+

- y7+

- y8+

- y9+

- y5+

- y4+

- y3+

- y2+

- y13+

- y12+

- y11+

- y10+

- y6+

- y7+

- y8+

- y9+

- y5+

- y4+

- y3+

**A  C  Q  E  Q  I  E  A  L  L  E  S  S  L  R**

**A  C  Q  E  Q  I  E  A  L  L  E  S  S  L  R**

- ICAT-13C9

- ICAT-13C0

- y3

- y4

- y5

- y6

- y7

- y8

- y9

- y10

- y11

- y12

- y13

- y2

- y3

- y4

- y5

- y6

- y7

- y8

- y9

- y10

- y11

- y12

- y13

**Cyclin D1**

**Cyclin D1**

**Identification of ICAT-labeled Peptides**

## B.

**81.0**

**82.0**

**83.0**

**84.0**

**85.0**

**Retention Time (min)**

**0**

**50**

**100**

**Relative Abundance**

**81.0**

**82.0**

**83.0**

**84.0**

**85.0**

**Retention Time (min)**

**0**

**50**

**100**

**Relative Abundance**

**Cyclin D1**

**Actin**

**S               P **

**B.**

**13****C****9****/****13****C****0**** ratio = 1.76**

**Densitometric ratio = 2.44**

## Validation of Quantitative Proteomic Data

**Validation of Quantitative Proteomic Data**

## Integration of Transcriptomic and Proteomic Data: 

**Integration of Transcriptomic and Proteomic Data: **
**From Global to Pathway to Protein**

## Bridging the Gap Between Discovery and 

**Bridging the Gap Between Discovery and **
**Hypothesis-Driven Biomarker Discovery**

## Biomarker Identification

**A major focus in proteomics is to leverage MS technology to identify biomarkers from human biofluids.**
**As with characterization of protein complexes, any sample preparation that can help target the desired biomarker is invaluable.**
**Minimizing the “background” greatly simplifies the uncertainty in the biomarker that is eventually identified.**

## Biomarker Discovery is Challenging!

**Samples can be difficult to obtain.**
**	****invasive collection**
**	****lack of retrospective samples**** **
**	**

**Non-standardized sample collection.**
**	****plasma vs. serum**
**	****effect of processing, storage, etc.**** **
**	**

**Potentially valuable biomarkers are diluted into a vast “sea” of biological background prior to sample acquisition.**
**	**

**Biomarker Discovery is Challenging!**

## Interstitial Cystitis 

**Interstitial Cystitis **

**Interstitial cystitis (IC) is a chronic, painful urinary bladder disorder.**
**It is characterized by thinning and ulceration of the bladder epithelial lining.**
**The etiology of IC is unknown and present diagnosis is performed through the exclusion of other conditions followed by a bladder biopsy under general anesthesia.**
**Approximately 1 million people in the United States suffer from IC. **

## Antiproliferative Factor 

**Antiproliferative Factor **

**An antiproliferative factor (APF) was previously described to be produced by the bladder epithelial cells of IC patients.**
**APF is found in both the urine and supernatant of explanted bladder epithelial cells of IC patients.**
**The presence of APF was first discovered in 2000, however, its structure as remained elusive.**
**Preliminary characterization of APF suggest it is a low molecular weight, relatively heat stable, peptide. **

## Isolation of Wild-type APF 

- Ion Exchange

- Hydrophobic Interaction

- Reversed-phase

- Ultrafiltration

**0**

**10**

**20**

**30**

**40**

**Retention Time (min)**

**0**

**100**

**Relative Abundance (%)**

**1**

**2**

**3**

**4**

**5**

**6**

**Isolation of Wild-type APF **

## Mass Spectrometry Analysis of Purified APF 

- 500

- 700

- 900

- 1100

- 1300

- 1500

- 1700

- 1900

***m/z***

- 0

- 50

- 100

- Relative Abundance

- 827.1

- 1482.8

**Mass Spectrometry Analysis of Purified APF **

## Determination of Correct APF Peptide Sequence

**541**

**549**

**Determination of Correct APF Peptide Sequence**

## Validation of Proposed APF Structure

**Validation of Proposed APF Structure**

## Comparative Activity of Synthetic and Wild-type APF

**40**

**60**

**80**

**100**

**20**

**Native APF**

**Native Mock**
**APF**

**Synthetic**
**APF**

**Synthetic**
**Peptide Alone**

**Percentage Decrease in**
** ****Live Cell Count**

**Comparative Activity of Synthetic and Wild-type APF**

## Experimental Trade-offs in Biomarker Discovery

**Experimental Focus**

**MS Data Acquisition**

**Bioinformatic Effort**

**Collection of a lot of MS data requires a lot of bioinformatic analysis but little sample preparation.  *****Validation is still a long way off.***

**Sophisticated or targeted sample preparation often requires the collection of little MS data and little bioinformatic analysis.  *****Validation is closer.***

**Experimental Trade-offs in Biomarker Discovery**

## Proteomics

**Protein Identification**
**Global Proteomic Mapping**
**Quantitative Proteomics**
**Global Phosphorylation Mapping**
**Biomarker Discovery**

**Proteomics**