Leveraging the simplicity of MALDI-TOF MS for rapid characterization

33212 - MALDI-TOF mass spectrometry
33212 - MALDI-TOF mass spectrometry:

Not just for clinical microbiology labs anymore
  Leveraging the simplicity of MALDI-TOF MS

Dr. Mari DeMarco
  Leveraging the power of MALDI-TOF MS

Dr. Tony Hu
  Leveraging the flexibility of MALDI-TOF MS

Dr. Yusheng Zhu
69th AACC Annual Scientific Meeting – San Diego, 2017

  Leveraging the simplicity of MALDI-TOF MS:

Rapid characterization of endogenous peptide and
protein isoforms
Mari DeMarco, PhD DABCC FACB

Clinical Chemist, St. Paul’s Hospital, Providence Health Care
Clinical Associate Professor, University of British Columbia
Department of Pathology and Laboratory Medicine
Vancouver, Canada
Email: mdmrco@mail.ubc.ca
Speaker Financial Disclosure Information
§  Grant/Research Support: None
§  Salary/Consultant Fees: None
§  Board/Committee/Advisory Board Membership: None
§  Stocks/Bonds: None
§  Honorarium/Expenses: None
§  Intellectual Property/Royalty Income: None

Acknowledgements
Dr. Emma Zheng Dr. Junyan Shi Grace van der Gugten
Postdoctoral Fellow Postdoctoral Fellow Assay Development Specialist
Pawan Dhaliwal Amy Nguyen Dr. Serena Singh

Undergrad Graduate student Postdoctoral Fellow
Terry Wong Dr. Swamy Ravipati Raha Masoudi

Undergrad Postdoctoral Fellow
Undergrad
Overview
Leveraging the simplicity of MALDI-TOF MS
MALDI-TOF MS Basics
Epitope mapping
Rapid characterization of peptide/protein mixtures
Quality control
MALDI-TOF MS
Matrix Assisted Laser Desorp0on/Ioniza0on-Time of Flight MS
Image: http://www.sigmaaldrich.com/technical-documents/articles/biology/custom-dna-oligos-qc-analysis-by-mass-spectrometry.html
MALDI-TOF MS
§  Initially developed for pure peptide

and protein solutions
§  Soft ionization technique

Overview
§  Broad mass range
§  High sensitivity
§  Most commonly used for qualitative

application
Characteristics of MALDI-TOF MS
Mass Range
•  500 Da – 100 kDa+
Sensitivity
•  1 μL of 100 fmol - 10 pmol
Mass Accuracy
Image: www.protein.iastate.edu/maldi.html
•  ±0.1% (1 Da per 10 kDa)
Sample preparation
1.  (Sample prep)

Workflow
2.  Apply sample
3.  Overlay with matrix

§  Select appropriate matrix
§  Co-crystalize with sample
§  Help desorb/ionize sample
Stainless steel 4.  Sample = sample-matrix-

target plate solvents-additives
Matrices for MALDI-MS

Sinapinic acid -Cyano-4-hydroxycinnamic 2,5-Dihydroxybenzoic acid
(SA) acid (CHCA) (DHB)
Proteins, Peptides, Peptides,

peptides, nucleotides, oligonucleotides,
lipids lipids oligosaccharides
Images: Sleno L, Volmer DA. Rapid Commun Mass Spectrom. 2005;19(14):1928-36
MALDI-TOF MS
sample delivery
Desorption/Ionization
Laser
•  Matrix ( )absorbs energy,
generating excited energy state
+ •  Localized heating causes micro-

+ explosion of material
+
+ •  Collisions with neutral sample
+ + facilitate charge transfer to/from
+ excited matrix molecules
+
+
+
•  Soft ionization technique
•  +ve ion mode: M à [M + H]+
•  -ve ion mode: M à [M - H]-
+ -
Time of Flight
Linear Mode
Source DriH region (flight tube) Detector
+
+
+ +
+
§  Small ions reach the detector before large ones
§  Time of flight: time required for ions to reach the detector
Time of Flight
Linear Mode
The mass-to-charge ratio of an ion is

proportional to the square of its drift time.
t = Drift time
m 2t 2 K L = Drift length
= 2 m = Mass
z L K = Kinetic energy of ion
z = Number of charges on ion
Time of Flight
Reflectron Mode
Source DriH region (flight tube)
+
+
+ +
+
+
V Reflectron
Detector (ion mirror)
Reflectron: a gridded ion mirror that subjects the ions to a

uniform repulsive electric field to reflect them.
MAL + DI + TOF + MS =
Spectrum
Overview
✔ MALDI-TOF MS Basics
Epitope mapping
Quality control
Epitope Mapping
•  Using MALDI-TOF MS to map epitopes of commonly

used commercial antibodies against ACTH
•  Used to develop plasma ACTH immunoprecipitation

—mass spectrometric methods including:
–  Qualitative MALDI-TOF MS method to characterize the
ACTH-ome
–  LC-MS/MS method to quantitate the plasma ACTH-ome

ACTH - Structure
SYSMEHFRWGKPV GKKRRPVKVYPNGAEDESAEAFPLEF
ACTH
(1-39)
-MSH (1-13) CLIP (18-39)
Residues 1-24 are sufficient for biological activity
Making use of well characterized

antibodies to build an IP method
•  Roche Elecsys ACTH reagent kit
(electrochemiluminescent immunoassay)
•  Capture antibody (biotinylated)
•  Streptavidin-coated magnetic particles

Image: Roche ACTH assay fact sheet
Epitope Mapping by
Immuno-MALDI-TOF MS
Zheng, Shi, Wong, Dhaliwal & DeMarco (2017) In prep.
MALDI-TOF MS
Incubate with Add streptavidin- Apply magnet, Elute & Dry &
biotinylated anti- coated magnetic remove collect ACTH resuspend in
ACTH antibody beads supernatant isoforms matrix
•  α-cyano-4-hydroxycinnamate matrix
•  MALDI-TOF MS (FDA cleared)

Epitope mapping of Roche Ab
•  Published ACTH epitopes:
SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF
Residues: 9-12 36-39
•  Which antibody belongs to which epitope?
4.7 Lin Internal Mass AccuracyTof2_mix

4.7 Lin Internal Mass AccuracyTof2_mix
Data: <Untitled>.1A4[c] 11 Jan 2016 17:38 Cal: Ecal_CHCA 4.7 Lin Internal Mass AccuracyTof2_mix
Data: <Untitled>.1A4[c] 11 Jan 2016 17:38 Cal: Ecal_CHCA
Shimadzu Biotech Axima Assurance 2.9.3.20110624: Mode Linear_SARAMIS_20140424, Power: 53, Blanked, P.Ext. @8330 (bin 130)
%Int. 0.0 mV Profile 49 Data: <Untitled>.1E1[c] 11 Jan 2016 17:21 Cal: 15 Aug 2012 3:14
%Int. 0.0 mV Profile 49
Synacthen
%Int. 227 mV Profile 100
100
100
100
80
80
80
60
60 SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF
60
40
40
Residues 1-24
40 MWT 2933.44 g/mol
20
20
20
0
%Int.
0
1276 mV[sum= 62502 mV] Profiles 1-49 Smooth Av50
Stock solution
0
Immunopreciptate (1 μM)
%Int. 1276 mV[sum= 62502 mV] Profiles 1-49 Smooth Av50
%Int. 216 mV[sum= 21565 mV] Profiles 1-100 Smooth Av50
2933.9576{r307}
100 2933.9576{r307} 2934.4431{r565}
100
Rela0ve intensity (%)
100
80
2934 m/z 2934 m/z
80 80
60
60 60
40
40 40
2949.7584{r227}
20 20
20 2955.9282{r87}
2917.3922{r79}
2917.3922{r79} 2978.1439{r51}
0 0
0
2800 2820 2840 2860 2880 2900 2920 2940 2960
2800 2980
2820 3000 3020 3040 3060 3080 3100
2800 2820 2840 2860 2880 2900 2920 2940 28402960 2860
2980 30002880 3020 2900
3040 2920
3060 3080 2940
3100 2960 2980 3000 3020 3040 3060 3080 3100
m/z m/z
m/z
m/z m/z
CLIP
Synthetic (Bachem) Residues 18-39 MWT 2465.7 g/mol
CLIP stock solution Immunoprecipitate

(1, 100, 400 μM)
Rela0ve intensity (%)
2464 m/z
m/z m/z
Using MALDI-TOF MS to epitope

map Roche Elecsys ACTH kit
9-12 36-39
Overview
✔ Epitope mapping
Quality control
Characterizing the plasma ACTH-ome
What ACTH fragments are found in plasma?
SYSMEHFRWGKPV GKKRRPVKVYPNGAEDESAEAFPLEF
ACTH
(1-39)
-MSH (1-13) CLIP (18-39)

Immuno-MALDI-TOF MS
MALDI-TOF MS
Immuno-MALDI-TOF MS
ACTH-ome identification
4.7 Lin4.7
Internal
Lin Internal
Mass Mass
Accuracy
Accuracy
Tof2_mix
Tof2_mix
Data: Data:
<Untitled>.3I1[c]
Shimadzu
<Untitled>.3I1[c]
Shimadzu
Biotech
Biotech
AximaAxima
%Int. %Int. 43 mV[sum=
43 mV[sum=
Human plasma pool (n = 15)
17 Oct172016
Oct 16:49
Assurance
4287 mV]
2016 16:49
Assurance
Cal: 15
Cal:
2.9.3.20110624:
Aug
152012
Aug 5:14
2.9.3.20110624:
4287 Profiles
mV] Profiles
2012 5:14
Mode Mode
1-1001-100
Linear_SARAMIS_20140424,
Smooth
Smooth
Linear_SARAMIS_20140424,
Av 50Av 50
Power:Power:
54, Blanked,
54, Blanked,
P.Ext.P.Ext.
@ 8330@(bin
8330130)
(bin 130)
2229.9448{r545} 8"26%
2229.9448{r545}
•  Multiple independent
100 100
95 95
90 90
85 85
80 80
plasma pools analyzed
75 75
•  Results compared/
70 70
65 65
Rela0ve%intensity%%%
combined to develop
60 60
55 55
50 50
45 45
40 40
9"39%or%8"38%
3509.8920{r734}
3509.8920{r734}
plasma ACTH-ome list
35 35
•  Notable for multiple N-

30 30
25 25
2"19%or%9"28%
terminal truncations
20 20
15 15 2258.0530{r571}
2258.0530{r571}
2"32%
10 10 2133.2631{r515}
2133.2631{r515} 8"30%or%9"32% 3624.9587{r763}
3624.9587{r763} 2"36%
2662.0264{r641}
2662.0264{r641}
4067.0995{r824}
4067.0995{r824}
5 5
0 0
Zheng, Shi, Wong, Dhaliwal & DeMarco. In prep.

1500 1500 2000 2000 2500 2500 3000 3000 3500 3500 4000 4000 4500 4500
m/z m/z
m/z%
Overview
✔ Epitope mapping
✔ Rapid characterization of peptide/protein mixtures
Quality control
MALDI-TOF MS for Quality Control
Internal standard for ACTH quantification by

LC-MS/MS
Human: SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF
Mouse/Rat: SYSMEHFRWGKPVGKKRRPVKVYPNVAENESAEAFPLEF
Immuno-MALDI-TOF MS
MALDI-TOF MS
MALDI-TOF MS for quality control

•  IS: Mouse/Rat synthetic peptide (Bachem)
•  MWT: 4582.23, HPLC Purity: > 95%
•  IP: 100 M stock solution
Rela0ve Intensity (%)
+2 +1
m/z
Using MALDI-TOF MS data to build a

plasma ACTH IP-LC-MS/MS method
•  Multiple reaction
monitoring (MRM)
method for plasma
3 (cps)
ACTH peptide
Intensity (cps)
fragments
Intensity x 10
•  HPLC-triple quadrupole
mass spectrometer
ACTH
1-39 •  Data from human
plasma pool
Time (min)
MALDI-TOF MS used to help

characterize the plasma ACTH-ome
•  We identified multiple abundant ACTH
fragments in plasma, documenting the
complexity of the plasma ACTH-ome
ACTH+
+
+
ACTH+
•  We identified discrepancies in ACTH-
+ ome immunoassay reactivity among
ACTH+ +
+ + different manufacturers
ACTH+
+
+
•  We identified isoforms with assay-
ACTH+
reactivity in the absence of biological
activity
Summary
• Sample & slide prep, instrumentation,
✔ Epitope mapping
• For troubleshooting complex cases & assay development
✔ Rapid characterization of peptide/protein mixtures

• Identification of the plasma ACTH-ome
✔ Quality control of standards

• Confirming purity of peptide solutions

  Leveraging the power of MALDI-TOF MS:

Quantitation of protein biomarkers using nanoparticles
Tony Y. Hu, PhD

Associate Professor
School of Biological and Health Systems Engineering
Virginia G. Piper Biodesign Center for Personalized Diagnostics,
The Biodesign Institute, Arizona State University
Email: tyhu@asu.edu
Absolute Quantification with MALDI-TOF MS
•  Mass spectrometry (MS) detection techniques for the detection and

quantitation of protein targets in circulation cannot achieve the sensitivity of
ELISA or RIA, without the selectivity of an immuno-affinity concentration step.
•  MRM can provide absolute quantitation of peptides and small proteins in

body fluids by means of MS/MS, but requires sophisticated instrumentation
and highly skilled operators.
•  The use of immuno-MALDI-TOF MS as a detection system is faster, suitable for

high-sensitive and high throughput analyses, and technically less sophisticated
than LC-ESI or triple quad MS-based systems.
•  The incorporation of stable-isotopically labeled peptides as internal standards

permits absolute quantitation.

Quantification of Circulating M. Tuberculosis

Antigens for Rapid Diagnosis and Real-time
Treatment Monitoring
Proc. Natl. Acad. Sci. U S A. 2017, 114(15):3969-3974
Tuberculosis (TB): Facts
An infectious disease caused by

Mycobacterium tuberculosis, an
airborne microbial pathogen.
One third of the world’s population

infected.
82% of those infected live in only 22

countries.
A leading killer for HIV-infected people.
Extremely difficult to detect at early or in

latency
Tuberculosis Diagnostics: Bottlenecks

•  Microbiologic techniques including sputum smear microscopy and Mtb
culturing.
-  low sensi7vity, moderate specificity and/or long turnaround 7mes;
-  Difficult to iden7fy pa7ents who expel liOle to no bacteria (e.g., HIV/TB co-
infected cohorts) or pa7ents with nonspecific symptoms (e.g., extrapulmonary
TB or pediatric TB).

•  GeneXpert does not address the issue of low sensi7vity due to the
dependence of bacterial load.
•  IFN-γ release assays (IGRAs) fail to dis7nguish ac7ve TB disease from

LTBI, due to persistent immunologic responses from long-lived human
memory T-cells to Mtb.
None of these techniques offers quantitative results.
Tuberculosis Diagnostics: The Highest Priorities
1.  Rapid and independent of bacterial isolation (culture-

free);
2.  Non–sputum-based test capable of detecting all forms of
tuberculosis via the identification of characteristic
biomarkers;
3.  Quantifiable and informative in detecting treatment
response.
Our Hypothesis - to Fill the Conceptual and Technical Gaps
•  CFP-10 and ESAT-6 are two TB virulence factors that dimerize to cause
immunopathogenic responses in the human host.
•  The presence of these two secretory an7gens is evidence of ac?ve Mtb, allowing
them to be ideal biomarker for disease diagnosis.
To identify Mtb-specific antigen markers

CFP-10 ESAT-6
a
b
*
*
*
To identify Mtb-specific antigen markers
CFP-10
ESAT-6
1 µm
100 100
1593.75 1900.95
with pSiNDs with pSiNDs
80 without pSiNDs 80 without pSiNDs
Relative Intensity (%)
60 60
40 40
20 20
0 0
1580 1585 1590 1595 1600 1605 1890 1895 1900 1905 1910 1915 m/z
m/z
Sharpening our knife

Method development
Traditional digestion CHCA No
a b CHCA+pSiNDs
CHCA+AuNPs
c enrichment
800 Microwave assisted 500 CHCA+Silica NPs Antibody

digestion CHCA+Graphene 1200
*** conjugated
700 *** pSiNDs
***
Normalized Intensity
1000
400 Antibody
600 conjugated
500 800 AuNPs
300
*** *** ***
400 600
300 200
400
200
100 200
100
0 0 0
CFP-10 ESAT-6 CFP-10 1593.75 ESAT-6 1900.95 CFP-10 ESAT-6
1593.75 1900.95 1593.75 1900.95
SiO2
d e f
Si
SiO2
Si
SiO2
1 µm 2 nm
Absolute quantification of Mtb antigens
4.5 CFP-10
a b 4
0.4
ESAT-6
Target Fragment Intensity
3.5 0.2
3
/ Its Isotope
2.5 0
2 0 1 2
1.5
1
0.5 R² = 0.99
0
0 5 10 15 20
c Concentration (nM)
100 1603.60 100

1910.80
75 75
50 50
1593.75 1900.95
25 25
0 0
1592 1596 1600 1604 1608 1898 1902 1906 1910 1914
Patient # 1243.01
100
Mtb Culture: positive
Clinically confirmed*
75
50 CFP-10 Control # 1139.04

ESAT-6
25
0
1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 m/z
Preclinical Valida?on
Diagnos?c Performance of ND-MS Assay
Performance of ND-MS in Culture-nega?ve Pa?ents
Preclinical Validation
Pulmonary TB n=76
Extrapulmonary TB n=19
Healthy control n=96
Pneumonia n=40
NTM n=24
CFP-10
ESAT-6
0 18nM
Active TB vs. LTBI

Pulmonary TB n=76
Extrapulmonary TB n=19
LTBI n=31
0 18nM
CFP-10
HIV+/LTBI n=30 ESAT-6
Diabetes/LTBI n=18
Diagnosis of TB in HIV
ü  WHY is it difficult to diagnose TB in HIB NanoDisk-MS may adversely skew
infected pa9ents the results of indirect host response-
•  Frequently nega7ve sputum smears based immunoassays.
•  Atypical radiographic findings ***
160
•  Resemblance to other opportunis7c 80
pulmonary infec7ous pneumonia

circulating CFP-10 & ESAT-6 (nM)
80
Our results
Sum of concentration of
10
140
circulating CFP-10 & ESAT-6 (nM)
120 8
Sum of concentration of
60
40 6
20
20
4
15
2
10
5 0
0 TB+/HIV- TB+/HIV+
(n=95) (n=23)
TB+/HIV+ TB-/HIV+
(n=23) (n=23)
HTI pediatric data

Mtb Culture + Mtb Culture -
n=23 n=28
Healthy control
n=51
0 2nM
CFP-10
LTBI
ESAT-6
n=21
Iden?fica?on of ac?ve TB in pediatric cases. Circula7ng CFP-10 (red) and ESAT-6 (blue) were
quan7fied in sera collected from 51 pediatric pa7ents with ac7ve TB (23 Mtb culture-posi7ve
and 28 Mtb culture-nega7ve), 21 pediatric pa7ents with LTBI, and 51 pediatric healthy controls
(results are averaged from 3 individual tests of each serum sample). The color gradient
indicates an7gen concentra7ons from 0 to 2 nM.
Evaluation of TB Treatment Efficacy
Detec9ng pa9ents’ response to treatment in one week
Patient #: 4 Patient #:
4 218863 218815
3 3
2 2
CFP-10
1 1 ESAT-6
0
0
Sample Collections
Treatment Sample A Sample B Earliest clinical Clinical diagnostic

Patient ID
start date collection collection diagnosis technology
218863 6/14/2014 6/14/2014 6/18/2014 7/23/2014 MTCT

218815 6/11/2014 6/11/2014 6/19/2014 7/29/2014 MTCT
Performance in a longitudinal cohort
Adults
Pediatrics
Short-term Evaluation of anti-TB treatment efficacy.
Mtb antigen quantification in serum of prospectively enrolled adult TB subjects with

multiple early treatment time points.
Highlights
•  Detection and quantification of TB antigens correlates with
bacterial load and infection
•  Samples obtained through a relatively simple blood draw

rather than through invasive biopsy or expensive image re-
staging scans
•  Outperforms current proteomic technologies with low
detection limit of 1.0 fmol and a target isolation rate of 90%.
•  Substantially diminishes the rate of false-positives
•  Time saving and high-throughput
•  Bench-top MALDI-TOF MS version is now readily available

for clinical use
•  Nanodisks can be manufactured in bulk at very low costs
Strategic Plan
Optimize development a portable system
for identifying and quantifying TB
biomarkers
Pinch
HV Valve LIT 1 LIT2
Ion
LIT 1 Detector
m/z
LIT 2
MS/MS 5 Turbo 11-80 l/s
l/min Pump
Where our ideas comes from - Exosome Study

TB Diagnosis Dr. Eugene Koay (MD Anderson)
Dr. David Bernard (HMRI)
Dr. Edward Graviss (HMRI) Dr. Zhen Zhao (NIH Clinical Center)
Dr. Charles Mitchell (Univ. of Miami) Dr. Kenji Yokoi (HMRI)
P1041 Study Team
Dr. Steve Holland (NIH NIAID) Cardiotoxicity Study
Dr. Irini Sereti (NIH NIAID)
Dr. Adrian Zelazny (NIH Clinical Center) Dr. Edward Yeh (MD Anderson)
Dr. Anneke Hesseling (Stellenbosch Univ., Dr. Michael Fisch (MD Anderson)
South Africa) Dr. Ziding Feng (MD Anderson)
Dr. Elisabetta Walters (Stellenbosch Univ.,
South Africa) Tech Development
Cancer Biomarkers Dr.. Zheng Ouyang (Purdue
Univ.)
Dr. Li Ma (MD Anderson)
Dr. Xuewu Liu (HMRI)
Dr. Paul Chiao (MD Anderson)
Dr. Chris7ne Singer (Vienna Medical School)
Dr. Selina C. Kiang (Weill Cornell Medical College)
EBOLA Detec7on
Dr. Francisco Esteva (New York University) Dr. Lisa H. Cazares (USAMRIID)
Dr. Kathryn Hajjar (Weill Cornell Medical College)
Acknowledgement
Acknowledgement
John S. Dunn Foundation


  Leveraging the flexibility of MALDI-TOF MS:

Application to molecular genetics and toxicology
Yusheng Zhu, PhD, DABCC, FACB

Professor of Pathology and Lab Medicine
Medical Director, Clinical Chemistry
Co-Director, Pathology Core Reference Lab
Penn State University Hershey Medical Center
Email: yzhu@pennstatehealth.psu.edu
Overview
Leveraging the flexibility of MALDI-TOF MS
•  MALDI-TOF MS and nuclei acid analysis
•  MALDI-MS imaging
•  MALDI-TOF MS and mass cytometry

MALDI-TOF MS AND NUCLEI

ACID ANALYSIS
The Challenge of Nucleic Acid

Analysis by MS
n  Labile phosphodiester backbone and the
tendency for base cleavage from deoxyribose:
Difficult to volatilize
n  Affinity of the negatively charged backbone
phosphate groups for nonvolatile cation (Na,K)
adduction spreads the molecular ion signal
into several peaks: Decrease the S/N ratio and
molecular mass measurement accuracy
Doktycz et al. Anal Biochem 1995; Little et al. Proc Natl Acad Sci U S A 1995
The first nucleic acids

detected by MALDI
n  Mass of oligothymidylic acid d(pT)15
Karas et al. Trends in Analytical Chemistry 1990

Typical Workflow for MALDI-

TOF MS Based SNP Typing
Pusch et al. Pharmacogenomics 2002
Molecular Pathology
http://agenabio.com/wp-content/
uploads/2015/07/51-20061R1.0-
iPLEX-Application-Note_WEB.pdf
Advantages of MALDI-TOF MS
for SNP Typing
n  Measurement of an intrinsic property of
the analyte
n  High speed
n  Multiplex ability
n  Automation
n  Sensitivity and Accuracy
n  Platform technology for functional
genomics approaches
MALDI-TOF MS: functional

genomics approaches
MALDI-TOF MS for CFTR

genotyping
n  CF: caused by the mutations in both copies
of the gene for the cystic fibrosis
transmembrane conductance
regulator (CFTR) protein
n  Over 1500 other mutations can produce CF
n  ΔF508 accounts for 66–70% of CF cases
worldwide and 90% of cases in the United
States
MALDI-TOF MS for CFTR

genotyping
Braun et al. Clin Chem 1997

Beta-thalassemia-Multiplex
SNP Typing
n  Beta-thalassemia: a common hereditary
disease caused by the mutation of a
single gene
n  Approximately 200 different mutations
n  Large deletions are sometimes observed
Beta-thalassemia-Multiplex
SNP Typing
Liao et al. J Hum Genet (2005)
Blood Group Genotyping

n  328 recognized blood group antigens
n  Of which 284 are comprised within 30
blood group systems, have been
described on the molecular level
n  Most antigens differ from their
antithetic partner by Single Nucleotide
Polymorphisms (SNPs) in their genomic
sequence
Blood Group Genotyping
Gassner et al. Transfusion Medicine Reviews 2013
EGFR genotyping by
MALDT-TOF MS
n  Patients with NSCLC with EGFR–
activating mutations have excellent
response to EGFR TKIs.
n  But T790M mutation accounts for most
TKI drug resistance.
n  Direct sequencing and NGS
n  MALDI-TOF MS
Su, et al. J Clin Oncol 2012
EGFR T790M genotyping

by MALDI-TOF MS

EGFR: MALDT-TOF MS vs
NGS/Direct Sequencing
Maternal serum free placenta mRNA

amplification for Down syndrome
n  An extra chromosome of number 21

n  Maternal screening
n  Second trimester (16- to 18-week)
n  First-trimester (10- to 13-week)
n  Maternal serum free placenta mRNA
amplification
Maternal serum free placenta mRNA

amplification for Down syndrome
Lo, et al. Nat Med 2007

18
HBV-Multiplex SNP Typing

n  HBV variants frequently develop with
long-term antiviral therapy
n  Some of these variants confer drug

resistance
n  60 frequently observed HBV variants
HBV-Multiplex SNP Typing
Luan et al. Clin Chem., 2009
Microbiology
Fournier PE. Nature Review Microbiology, 2013, 11:574-585

MALDI-TOF MS IMAGING
Tissue Imaging
Aichler & Walch. Laboratory

Investigation (2015) 95, 422–431
Mainini et al. Methods Mol Biol. 2015

MALDI Imaging: from whole body

to subcellular level
Ait-Belkacem, Trends in Biotechnology 2012
Localization of cell organelles by

MALDI-TOF MS
Ait-Belkacem, Trends in Biotechnology 2012
Chemical analysis in fingermarks

using MALDI-TOF MS
Kaplan-Sandquist et al. / Forensic Science International 2014

Hair Drugs Distribution Based

on MALDI-TOF MS Imaging
Flinders, et al. Drug Test. Analysis 2015
Detection of Colorectal Cancer Liver

Metastasis by MALDI-TOF MS
Imaging
Thomas, et al. Anal. Chem. 2013
Tissue-resident lymphocytes
visualized by MALDI MSI
Holzlechner, J.
Proteome Res. 2017
Microbiology Imaging
Watrous JD & Dorrestein PC. Nature Review

Microbiology, 2011, 9:683-694
MALDI-TOF MS AND MASS

CYTOMETRY
Mass Cytometry
Bendall SC. et al. Trends in Immunolog, 2012, 33(7): 323-332

Mass Cytometry vs Flow Cytometry
n  High vs zero background;
n  Many different heavy metal isotopes

can be used (12 fluorescent dyes vs
>100 isotopes).
Detection of CD8+ T cells by

mass cytometry
Newell et al. Immunity. 2012
Thank You
yzhu@pennstatehealth.psu.edu

Leveraging the simplicity of MALDI-TOF MS for rapid characterization

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33212 - MALDI-TOF mass spectrometry

33212 - MALDI-TOF mass spectrometry:

Leveraging the simplicity of MALDI-TOF MS

Leveraging the power of MALDI-TOF MS

Leveraging the flexibility of MALDI-TOF MS

69th AACC Annual Scientific Meeting – San Diego, 2017

33212 - MALDI-TOF mass spectrometry:

Leveraging the simplicity of MALDI-TOF MS:

Mari DeMarco, PhD DABCC FACB

69th AACC Annual Scientific Meeting – San Diego, 2017

Speaker Financial Disclosure Information

§ Grant/Research Support: None

§ Salary/Consultant Fees: None

§ Board/Committee/Advisory Board Membership: None

§ Intellectual Property/Royalty Income: None

Pawan Dhaliwal Amy Nguyen Dr. Serena Singh

Terry Wong Dr. Swamy Ravipati Raha Masoudi

Rapid characterization of peptide/protein mixtures

§ Initially developed for pure peptide

§ Soft ionization technique

§ Most commonly used for qualitative

• 1 μL of 100 fmol - 10 pmol

1. (Sample prep)

3. Overlay with matrix

§ Co-crystalize with sample

§ Help desorb/ionize sample

Stainless steel 4. Sample = sample-matrix-

Matrices for MALDI-MS

Proteins, Peptides, Peptides,

Images: Sleno L, Volmer DA. Rapid Commun Mass Spectrom. 2005;19(14):1928-36

+ • Localized heating causes micro-

§ Small ions reach the detector before large ones

§ Time of flight: time required for ions to reach the detector

The mass-to-charge ratio of an ion is

Reflectron: a gridded ion mirror that subjects the ions to a

Rapid characterization of peptide/protein mixtures

• Using MALDI-TOF MS to map epitopes of commonly

• Used to develop plasma ACTH immunoprecipitation

– LC-MS/MS method to quantitate the plasma ACTH-ome

-MSH (1-13) CLIP (18-39)

Residues 1-24 are sufficient for biological activity

Making use of well characterized

• Capture antibody (biotinylated)

• Streptavidin-coated magnetic particles

• MALDI-TOF MS (FDA cleared)

Epitope mapping of Roche Ab

• Published ACTH epitopes:

• Which antibody belongs to which epitope?

4.7 Lin Internal Mass AccuracyTof2_mix

CLIP stock solution Immunoprecipitate

Using MALDI-TOF MS to epitope

Zheng, Shi, Wong, Dhaliwal & DeMarco (2017) In prep.

Rapid characterization of peptide/protein mixtures

Characterizing the plasma ACTH-ome

What ACTH fragments are found in plasma?

-MSH (1-13) CLIP (18-39)

• Notable for multiple N-

Zheng, Shi, Wong, Dhaliwal & DeMarco. In prep.

✔ Rapid characterization of peptide/protein mixtures

MALDI-TOF MS for Quality Control

Internal standard for ACTH quantification by

MALDI-TOF MS for quality control

Using MALDI-TOF MS data to build a

  Leveraging the simplicity of MALDI-TOF MS

  Leveraging the power of MALDI-TOF MS

  Leveraging the flexibility of MALDI-TOF MS

  Leveraging the simplicity of MALDI-TOF MS:

§  Grant/Research Support: None

§  Salary/Consultant Fees: None

§  Board/Committee/Advisory Board Membership: None

§  Intellectual Property/Royalty Income: None

§  Initially developed for pure peptide

§  Soft ionization technique

§  Most commonly used for qualitative

•  1 μL of 100 fmol - 10 pmol

1.  (Sample prep)

3.  Overlay with matrix

§  Co-crystalize with sample

§  Help desorb/ionize sample

Stainless steel 4.  Sample = sample-matrix-

+ •  Localized heating causes micro-

§  Small ions reach the detector before large ones

§  Time of flight: time required for ions to reach the detector

•  Using MALDI-TOF MS to map epitopes of commonly

•  Used to develop plasma ACTH immunoprecipitation

–  LC-MS/MS method to quantitate the plasma ACTH-ome

•  Capture antibody (biotinylated)

•  Streptavidin-coated magnetic particles

•  MALDI-TOF MS (FDA cleared)

•  Published ACTH epitopes:

•  Which antibody belongs to which epitope?

•  Notable for multiple N-

  Leveraging the power of MALDI-TOF MS:

§  Grant/Research Support: None

§  Salary/Consultant Fees: None

§  Board/Committee/Advisory Board Membership: None

§  Intellectual Property/Royalty Income: None

•  Mass spectrometry (MS) detection techniques for the detection and

•  MRM can provide absolute quantitation of peptides and small proteins in

•  The use of immuno-MALDI-TOF MS as a detection system is faster, suitable for

•  The incorporation of stable-isotopically labeled peptides as internal standards

•  IFN-γ release assays (IGRAs) fail to dis7nguish ac7ve TB disease from

1.  Rapid and independent of bacterial isolation (culture-

•  Samples obtained through a relatively simple blood draw

•  Substantially diminishes the rate of false-positives

•  Time saving and high-throughput

•  Bench-top MALDI-TOF MS version is now readily available

•  Nanodisks can be manufactured in bulk at very low costs

  Leveraging the flexibility of MALDI-TOF MS:

§  Grant/Research Support: None

§  Salary/Consultant Fees: None

§  Board/Committee/Advisory Board Membership: None

§  Intellectual Property/Royalty Income: None