Essential Elements of MS …

Data Analysis

Steve Stein
MS Data Center
Chemical and Biochemical Reference Data Division
Materials Measurement Lab
NIST

Material Measurement Lab

 Data Analysis
• Data
– MS: Qualities, behavior, formats
– LC/MS: Complexity and dimensions

• Analysis
– Algorithms: Mostly Proprietary
– Identity: How to Express Confidence

Material Measurement Lab

 MS Data
• Single stream of ions vs. time
– Detector: Electron multiplier or Ion image current
– A destructive ‘spectroscopy’

• Two time ‘domains’

– Time converted to m/z
– Time converted to chromatographic component

• Two ‘peaks’
– m/z and Chromatographic

• m/z from time, Intensity from current

• Charge (z) from isotope spacing

Material Measurement Lab

MS Signals vs Time
Noise

Intensity

Noise  K noise Intensity

Material Measurement Lab

 Mass Spectra: Qual/Quan
• Mass (m/z): Qualitative
– Measure chemical formula
• Elemental composition
– Provides Identification
– Accuracy depends on instrument class not ion source

• Intensity: Quantitative
– MS1: variable
– MS2: relative intensities may be reproducible
– Variable, depends on ion source

Material Measurement Lab

 2 Varieties of ‘Mass Spectra’
• MS1
– Intact ions (‘evaporate’ from solution, mostly)

• MS2
– Fragments of ions – depends on structure
– MS/MS, Tandem MS, MSn, PSD, …
– Wide or Narrow m/z range

Material Measurement Lab

 MS1
• Ion Profile from the ‘ion source’
• Variable : ‘matrix effects’ and source instability
• ESI, MALDI, ‘Ambient Methods’
• Proteins and Their Fragments
– Resolved 13C Isotopes (< 30 kD, 250 AA residues)
• Benefits from resolution and sensitivity
– Unresolved Ions ( > 30 kD, lower if heterogeneous)
• Benefits from sensitivity only

Material Measurement Lab

 MS2 – Wide precursor m/z range

• Multiple ions co-fragment

– In-source (all ions fragment)
– MSE, m/z range, Wide Band Ion Trap, …

• Different ions require different energies to

fragment
– m/z, charge state, structure

Material Measurement Lab

 MS2 – Select Individual Ions
• Energy Dependence
– Strong: Collision Cell (Beam-Type, QQQ) – Ions gain internal energy as decelerated
– Weak: Ion Trap – Ions heated in small increments until they fragment
– Fixed: Post-source decay (MALDI)– like Collision Cell but cannot vary energy

• Molecular ‘fingerprints’ for Individual Ions

– Determined by chemical structure
– Save in Library

• Predictability
– Small molecules (organic molecules) - NO
– Peptides, including glycopeptides – Yes

• Proteins and large fragments (research)

– Small fragments will be isotopically resolved

Material Measurement Lab

Mass Spectra are Reproducible

O’Neal et al.
Anal. Chem.
1951

NIST
2012

A mass spectrum is a property of an ion

Material Measurement Lab

 Collision
Energy Dependence Energy
Setting
Gly-3_NGA2-200x-HCD-5to55 #967 RT: 6.09 AV: 1 NL: 1.44E5
T: FTMS + c NSI d Full ms2 678.22@hcd30.00 [100.00-1370.00]
678.2228
100
2+ 30
R elative A bu nda nce

80 [M+H+K]
60

40
576.6827
20
204.0864 495.6573 991.2909
343.5973 394.1171 626.1581 829.2382 931.2584 1151.1477 1253.5557
0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z
Gly-3_NGA2-200x-HCD-5to55 #968 RT: 6.10 AV: 1 NL: 4.62E4
T: FTMS + c NSI d Full ms2 678.22@hcd35.00 [100.00-1370.00]
678.2226
100
Relative Abundance

80 576.6829
35
60
526.6601 695.3120
40
204.0863 495.6567
394.1174 991.2914
20 220.8554 343.5934
626.1591 829.2395
283.5729 769.2132 931.2676 1116.7522 1245.1434
0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
Gly-3_NGA2-200x-HCD-5to55 #969 RT: 6.10 AV: 1 NL: 2.09E4 m/z
T: FTMS + c NSI d Full ms2 678.22@hcd40.00 [100.00-1370.00]
138.0547
100
Relative Abundance

80

60
204.0863 394.1176
576.6827
40
283.5727
466.1385
40 495.6571 626.1594 991.2908
343.5940 678.2214 788.2122
20 728.1912 829.2380
931.2665
242.0537 1032.2496
0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z

Material Measurement Lab

Resistance to
fragmentation for
2500 tryptic
peptide ions

V1/2
product = precursor
abundance

QQQ Results for 25

Protein Digests

JASMS 2009 20, p.469

Material Measurement Lab

 Diving into the Data
RT: 11.91 - 81.24
100 NL: 1.92E6
Base Peak F: ITMS + c
ESI Full ms
95 [300.00-2000.00] MS
NCI_study2_021607_
90 sample1B33_vial_01

85

80

75

70

NCI_study2_021607_sample1B33_vial_01 RT: 20.00 - 80.00 Mas s: 300.00 - 1800.00 NL: 1.92E6 F: ITMS + c ESI Full ms [300.00-2000.00]
65 1800

60
1700
Relative Abundance

55

1600
50

45 1500

40
1400
35

1300
30

25 1200

20 6
1100 10

m/z
15

1000
10

5
Peptide
900

MS1 Maximum Intensity

0
15 20 25 30 35 40 45
Time (min)
50 55 60 65 70 75 80 800
Not Identified
700
5
10 Not Sampled
600

Chromatogram 500

400

4
300 10
20 25 30 35 40 45 50 55 60 65 70 75 80
Time (min)

“Ion Map” 3
10
20 30 40 50 60 70 80
Retention Time (min)

Ion Clusters

Material Measurement Lab

 Signals From Various Sources

noise
peptides
80
recurrent/noID
unmatched
unsampled
60
Intensity

40

20

0
10 20 30 40
Retention Time/s

Tryptic digest of egg white

Material Measurement Lab
Ion Components in a Protein Digest

Material Measurement Lab

 Signal Variability
Relative Peptide Ion Intensities In Successive Runs
100

10
Relative Intensity

0.1
0 50 100
RT/min
Yeast Tryptic Digest
Piening B.D. et al J. Proteomics Res. 2006, 5, 1527

Material Measurement Lab

 Successive Runs & First/Last Pair
100

10
Relative Intensity

0.1
0 50 100
RT/min

Yeast Tryptic Digest

Piening B.D. et al J. Proteomics Res. 2006, 5, 1527

Material Measurement Lab

 Huge Differences Between Labs

10 10
Intensity Ratio

Intensity Ratio
1 1

0.1 0.1
25 50 75 25 50 75
RT/min RT/min

Same Sample, Black: Successive Runs NIH/NCI/CPTAC

Instrument, Method Blue: Other Lab Interlab Studies, 2010

Material Measurement Lab

 Raw Data
• Data stored permanently on disk
– Is filtered from original signal – intensity vs. time
• Converted and stored as intensity vs m/z
• Classes of output
– Profile data – time to m/z only
– ‘Centroiding’ – identifies and adds signals for individual m/z peaks
– Files are big and growing in size: 500 MB / run not uncommon
• Inconvenient for Internet
• Title 21 CFR part 11 – keep for inspection with software?
– Web-based, raw data repositories struggling
• Size and annotation are major problem

• Each vendor has its own format(s)

– Access requires vendor involvement
• Application Programming Interface
• Translator to Standard Format (mzML – proteomics driven)
– Third party software is limited – for experts only
• Proprietary algorithms

Material Measurement Lab

 Data Analysis Software
• Proteomics Based
– Bottom Up (Infer Protein Structure from Fragments)
• X!Tandem, OMSSA, … (Public) MASCOT, Sequest (Proprietary)…
– Complex software packages – ‘validation’
– Protein ‘assembly’, finds only what you are looking for
• Specialized Biotherapeutics software (how it works)
– Other peptides – disulfides
– Epitope mapping – H/D exchange

– MW from charge state envelopes (‘deconvolution’)

• Black box: Heterogeneity & signal strength effects
– Top Down
• Prosite by Kelleher’s group

• Compare Data Files

– “Biomarker Discovery” – case/control
– Sample tagging (mass label on each sample)

Material Measurement Lab

 thresholds

Confirmed

Probably
right

Probably wrong Probably right

Material Measurement Lab

 peptide.nist.gov

NISTMSQC: Library/Quality Metrics for LC-MS/MS data

“Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in
Proteomics Analyses”, Molecular & Cellular Proteomics, 9, 225, 2010

Material Measurement Lab

 Peptide Library Spectrum Search

Spectrum
List Query
Spectrum

Score
Histogram

Hit
Library
List
(Consensus)
Spectrum

Will Hold Spectra of Biologic Drug Components

Material Measurement Lab
NIST Library Pipeline
Erbitux Heavy Chain - trypsin 2 hr

No Alkylation
high T Semi Tryptic
Missed Cleavage
Tryptic

urea

100 200 300 400

AA Position
Material Measurement Lab
 Final Thoughts
• Masses and identities can be determined with amazing
and increasing accuracy
– Just in time identities of biologic drug materials

• Sensitivity is increasing, but so is variability

– High sensitivity needed for HCP, contaminants
– Not for structure elucidation

• Proprietary software/formats dominate

– Changes with time and instrument

Material Measurement Lab