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The CHROMacademy Essential Guide Webcast:
The analysis of post-translational modifications using LC-MS/MS
Thursday 22nd August 2013

In this session, Dr Duncan Smith  and Dr John Griffiths (MS-Insight Ltd and Manchester University) explain the principles behind the analysis of a number of key post-translational modifications (PTMs) using LC-MS/MS techniques.  They will begin with a brief overview of bottom-up proteomics, explaining how proteins are converted to peptides, which are then analysed using electrospray LC-MS/MS.  They will move on to talk about the various MS scanning techniques, which may be used for PTM analysis. Next, they will discuss in turn, how phosphorylation, acetylation, ubiquitination and SUMOylation may be analysed using mass spectrometry, drawing upon examples from their own research at the University of Manchester. Finally, they will give their own opinions on the ways in which they see the future of PTM analysis heading.

Topics covered include:

  • What is proteomics and why are post-translational modifications important?
  • How are proteins identified using LC-MS/MS?
  • Utilizing different MS scanning functions to enable PTM detection
  • How to detect both global and targeted phosphorylation events.
  • Approaches to acetylation studies
  • How non-classical tryptic proteolysis of SUMOylated proteins enables their facile detection
  • Novel approaches for ubiquitination and SUMO determination
  • The future – combinatorial PTM detection with quantification?

Who Should Attend:

  • Scientists engaged in the field of proteomics
  • Anyone interested in the analysis of post-translational modificatons by LC-MS/MS
  • Mass spectrometrists who would like to keep up to date with the latest developments in PTM analysis
  • Biologists with questions to answer regarding the post-translational status of their proteins of interest
  GC-MS/MS


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The CHROMacademy Essential Guide Webcast:
The analysis of post-translational modifications using LC-MS/MS

Dr Duncan Smith  and Dr John Griffiths (MS-Insight Ltd and Manchester University) explain the principles behind the analysis of a number of key post-translational modifications (PTMs) using LC-MS/MS techniques.  They will begin with a brief overview of bottom-up proteomics, explaining how proteins are converted to peptides, which are then analysed using electrospray LC-MS/MS.  They will move on to talk about the various MS scanning techniques, which may be used for PTM analysis. Next, they will discuss in turn, how phosphorylation, acetylation, ubiquitination and SUMOylation may be analysed using mass spectrometry, drawing upon examples from their own research at the University of Manchester. Finally, they will give their own opinions on the ways in which they see the future of PTM analysis heading.

Topics covered include:

  • What is proteomics and why are post-translational modifications important?
  • How are proteins identified using LC-MS/MS?
  • Utilizing different MS scanning functions to enable PTM detection
  • How to detect both global and targeted phosphorylation events.
  • Approaches to acetylation studies
  • How non-classical tryptic proteolysis of SUMOylated proteins enables their facile detection
  • Novel approaches for ubiquitination and SUMO determination
  • The future – combinatorial PTM detection with quantification?

  ask the CHROMacademy experts

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Marc R. Wilkins is an Australian scientist who is credited with the concept of the proteome.1  Wilkins coined the term proteome in 1994 while studying for his PhD.2  This was a generalisation of the concept of the genome encompassing the set of all proteins that can be produced through the genome, through alternative splicing and post-transcriptional modification of messenger RNA.3,4  In the first book on proteomics in 1997 Marc Wilkins defined proteomics as: ‘Proteomics is the study of the entire protein complement of an organism.’4

The evolution of proteomics has developed into a field focused on the analysis of complex protein mixtures with 2D electrophoresis and/or mass spectrometry.  Proteomics is now dominated by the use of LC-MS/MS for the analysis of proteins.  The term ‘proteomics’ is now colloquially used to describe any protein analysis approach involving MS analysis.

Publications on Proteomics

Since its inception in 1997 the field of proteomics has seen an exponential rise in research output; from only 1 book in 1997 to over 5000 unique publications on the subject in 2012 (Figure 1).

 
Figure 1:  Number of proteomics publications from 1997 – 2012.
 
 

Cellular Signalling Cascades

Nearly every biological process is regulated by cellular signalling pathways, all of which rely on dynamic post translational modifications (Figure 2).  These post translational modifications are essential for the proteins to transmit their biological information, e.g. signals downstream of the TNFα (tumour necrosis factor alpha) receptor are reliant on both phosphorylation and ubiquitination of component protein molecules to transmit signals from the cell surface to the nucleus.  The critical role of such modifications simply cannot be overstated in the biological world.

 

Figure 2:  Cellular signalling cascades.5

 
 

Basic Principles of the LC-MS/MS Analysis of Peptides

The protein sample, be it a single protein or an entire proteome, is first digested into peptides in ‘bottom up’ proteomics research (Figure 3).  The proteolytic peptide products are much more amenable to successful analysis by liquid chromatography and tandem mass spectrometry than their intact protein counterparts.  These peptides are then analysed by LC-MS/MS and the resulting interrogation is by a database search of the appropriate protein sequences.

 

Figure 3: Schematic illustrating the steps involved in ‘bottom up’ proteomics research.

 
 

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All database search approaches are based on in silico prediction of a peptides characteristics followed by comparing this to the measured characteristics in the mass spectrometer.  Therefore, if we take the human protein sequence database and theoretically digest it with an enzyme with high specificity, the result is a list of peptide sequences (Figure 4).  From these sequences we can obtain elemental composition, and hence, theoretical mass.

 

Figure 4:  Peptide sequence.

 

Collisional-induced dissociation (CID) within the MS system results in preferential cleavage of the amide bond in the peptide, resulting in tandem mass spectra rich in either y type ions (when charge is retained on the C-terminus) or b type ions (if charge retention is on the N-terminus, Figure 5).

 

Figure 5:  Potential fragments of a peptide sequence produced in tandem MS.

 

Each of these theoretical fragment ions has an individual elemental composition and mass.  Therefore, before we have run any kind of mass spectrometry experiment we can predict highly specific potential masses for both the intact peptide and its potential fragments.

 
 

In Silico Peptide Analysis

A schematic representation of the in silico process of going from a protein sequence database to a final matrix of every peptide component is shown (Figure 6).  The matrix contains theoretical mass information for the intact peptide and its potential fragments.

 

Figure 6: Process of moving from a protein sequence database to a mass matrix of every peptide component.

 
 

Tandem MS for Protein Identification

Following the in silico analysis of the peptides characteristics, subsequent LC-MS/MS experiments revolve round the concept of collecting MS and MS/MS data throughout an LC separation.  The MS component collects m/z information (hence, mass can be inferred if z is detected) of the intact peptide components.  The population of ions representing any given peptide can be isolated, induced to fragment (in CID, either by collision with an inert gas or by resonant excitation) followed by mass analysis of the fragment ions.  This tandem mass spectrometry data can then be deconvoluted to generate an empirical matrix of masses both of the intact peptide and its detectable fragments (Figure 7).  The database search is effectively a statistical tool to assess the suitability of a match between a theoretical peptide matrix and that of an empirical matrix.

 

Figure 7:  MS spectra of the intact peptide (left).  Tandem MS/MS spectra of peptide fragment ions (middle).  MS matrix obtained (right).

 
 

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Phosphorylation

Phosphorylation of serine (S), threonine (T), and tyrosine (Y) is a prevalent post translational modification and occurs on more than one-third of all cellular proteins.13  Aberrations in these modifications may result in diseases such as cancers.  These modifications are often difficult to detect by mass spectrometry due to their low abundance, low stoichiometry, poor ionization, and complications of collision induced dissociation (CID) fragmentation.  Therefore, we require smart methods and/or enrichment to allow these aberrations to be detected.

Targeted mass spectrometric workflows can be used for the sensitive identification of post translational modifications (Figure 8).  Here the protein of interest is selected and an in silico digestion is carried out.  Multiple reaction monitoring (MRM) is used and the optimal fragmentation conditions are created and exported to the mass spectrometer; from here the instrument is able to carry out an MRM survey scan to detect potential modified peptides.  The strongest precursor ion above the set threshold is then selected and subjected to MS/MS resulting in the peptide sequence being obtained.  This approach is termed multiple reaction monitoring initiated detection and sequencing (MIDAS) and is more sensitive than other methodologies such as neutral loss scanning or precursor ion scanning. 

 

Figure 8:  MIDAS workflow.

 
 

The Cdc2 peptide (IGEGTYGVVYK, 1184.6 Da) has been subjected to a MIDAS study to identify the phosphorylation sites.  Table 1 contains transition information for the different forms of this phosphopeptide along with Q1 m/z, Q3 m/z, and collision energy. 

 
Q1 Q3 Amino Acid Sequence Fragment Type Fragment Mass Charge Collision Energy
673.2 624.2 IGEGpTpYGVVYK Loss of 49.0 +2 24.1
673.2 216.0 IGEGTpYGVVpYK Fragment 216.0 +2 60.0
633.2 584.2 IGEGpTYGVVYK Loss of 49.0 +2 22.3
633.2 216.0 IGEGTpYGVVYK Fragment 216.0 +2 60.0
449.1 416.5 IGEGpTpYGVVYK Loss of 32.7 +3 16.8
449.1 216.0 IGEGTpYGVVpYK Fragment 216.0 +3 60.0
422.5 389.8 IGEGpTYGVVYK Loss of 32.7 +3 16.1
422.5 216.0 IGEGTpYGVVYK Fragment 216.0 +3 60.0

Table 1:  Phophorylation studies.  MRM settings for Cdc2 peptide, IGEGTYGVVYK, 1184.6 Da with 2 potential phosphorylation sites and +2, +3 charge states.

 
 

Figure 9 shows a schematic of the detection of an MRM transition of the first phosphopeptide in Table 1 (IGEGpTpYGVVYK).  All peptide ions eluting from the LC column arrive at Q1.  Only transmission of m/z 673.2 is facilitated at this point in time, hence, only that ion is transmitted successfully into the collision cell.  Subsequently, this ion is fragmented by CID, but only the products of the ion m/z 624.2 are transmitted to the detector by Q3.  A signal is therefore only detected if a precursor ion of m/z 673.2 is present AND it fragments to give a product ion of m/z 624.2.  The process of precursor m/z to product m/z detection is termed a transition. In this example, an MRM transition of 673.2/624.2 is detected above a switching threshold (Figure 10).

 

Figure 9:  Schematic of an MRM transition for phosphopeptide IGEGpTpYGVVYK.

 
 

Figure 10:  Detected MRM transition of m/z 673.2/624.2.

 
 

The detection of this MRM transition above the set threshold is then used to trigger the data dependent acquisition of a full product ion spectrum for the precursor at m/z 673.2.  It is this product ion spectrum that contains the analytical information to confirm the sequence of the peptide and its site(s) of phosphorylation (Figure 11).

 

Figure 11:  Product ion spectrum of precursor ion m/z 673.2.

 
 

Figure 12 and 13 show the product ion spectra obtained from MRM of α-Casein, CDC13, and CDC2.

 

Figure 12: MS/MS spectrum of α-Casein S1.  10 fmol on column.

 
 

Figure 13:  Product ion spectrum of CDC13 and CDC2.

 
 

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Comprehensive post translational modification (PTM) analysis is more demanding than protein identification due to the greater protein sequence coverage that is required.  Specific proteases invariably do not produce digestion products amenable to high sequence coverage.  This enzyme ‘specificity’ is often considered as a strength, however, it is a key weakness too as a significant proportion of a protein’s sequence remains ‘invisible’ in a typical LC-MS/MS experiment.  This is dependent on the physiochemical nature of the particular peptide sequence.  An approach is needed to facilitate maximum coverage.  The use of using different ‘relaxed specificity’ enzymes is attractive for coverage and PTM analyses.14,15

Independent digestion of the proteins Fetuin and Rst2, with Trypsin, Elastase, Lys-N, and an Elastase/Lys-N combination demonstrates that trypsin digestion alone does not facilitate high sequence coverage.  Digestion with Elastase and LysN followed by no specificity database searches result in significantly greater sequence coverage than with trypsin alone.  The sequence coverages obtained from Elastase and LysN are complimentary.  In the Rst2 example, we are able to achieve sequence coverage of almost 90%, whereas, trypsin was only able to sequence about 15% of the molecule.  The independent digestion of Pom1 with Elastase and LysN demonstrates an ability to obtain high sequence coverage without any trypsin digestion.  The data demonstartes the limitations of trypsin proteolysis on sequence coverage.

 

Figure 14:  Enzyme digest coverage in ‘no specificity’ searches.

 
 

Comprehensive Phosphomapping of Wee1

Elastase, Trypsin, and Lys-N have been utilised in the digestion of Wee1 to allow for comprehensive phosphomapping (Figure 15).

 

Figure 15:  Strategy to comprehensively map phosphorylation sites on Wee1.

 
 

An LTQ-Orbitrap hybrid is typically used to acquire high resolution/high accuracy MS data in FT and low resolution/low resolution CID MSMS data in the IT (Figure 16).  The CID process is achieved by resonant excitation.  There is often complication of phosphorylation on serine (S)/threonine (T) residues.
The predominant loss of H3PO4 by beta elimination leaves MS/MS spectrum less analytically useful (Figure 17).

 

Figure 16:  Schematic of an LTQ-Orbitrap hybrid.

 
 

Figure 17:  Peptide CID (top).  pS/T peptide CID with neutral loss of H3PO4 (bottom).

 
 

Multistage activation (MSA) can be employed to further fragment products formed from the neutral loss of H3PO4
These multistage activation CID MS/MS spectra of pS/pT containing peptides are generally far more analytically useful than their CID counterparts (Figure 18).

 
Figure 18:  pS/T peptide MSA CID (top).  MSA (Jab and Uppercut) of neutral loss peptide (bottom).
 
 

The workflow delivered high sequence coverage of Wee1 (Figure 19).  94% sequence coverage was achieved with the workflow, with 34 distinct sites of phosphorylation confirmed.  Conversely, with trypsin alone less than 30% sequence coverage was achieved with only 8 confirmed sites of phosphorylation.

 

Figure 19:  Phosphorylation of Wee1 protein.

 
 

Global Phosphopeptide Enrichment

In global studies enrichment of phosphopeptides is key.  There are many approaches; however, the most generic is the use of titanium dioxide SPE. 
Figure 20 and table 2 show a representative schematic and the conditions employed in TiO2 SPE.

 

Figure 20:  Schematic of phosphopeptide enrichment via TiO2 SPE.

 
Buffer Composition
Load 80% ACN, 5% TFA, 1M Glycolic acid
Wash 1 80% ACN, 1% TFA
Wash 2 20% ACN, 0.1% TFA
Elute 1% ammonia

Table 2:  TiO2 SPE conditions for phophopetide enrichment.

 
 

Acetylation

Lysine acetylation is a reversible post translational modification that can occur on proteins involved in the regulation of diverse cellular processes, including mitochondrial functions (Figure 21).6,7  Identification of acetylation PTMs can be achieved through targeted approaches; (i) identification of an m/z 126.1 diagnostic fragment ion which corresponds to the induced fragmentation of the acetylated lysine immonium ion (m/z 143.1) via NH3 loss, (ii) the use of precursor ion scanning, and (iii) utilisation of MIDAS workflows.  Global analysis can also be used with the use of antibody based enrichment of peptides carrying acetyl lysine.  This would typically involve the digestion of an entire proteome followed by immunoprecipitating the peptides of interest for further LCMS/MS analysis. 

 

Figure 21:  Lysine acetylation.

 
 

Figure 22 shows data from the LCMS/MS acquisition of a digest of acetylated bovine serum albumin (BSA) in an excess of 6 protein mix digest.  Panel (a) shows the Total Ion Current (TIC).  Panel (b) is less complex as it uses precursor ion scanning at m/z 126.1.  Panel (c) is less complex still as it shows the TIC of an MRM scan as part of the MIDAS routine.  The use of precursor ion scans and MRMs seriously decomplexes the data, allowing for the focused analysis of only those analytes likely to carry acetyl lysine.

 

Figure 22:  Total ion chromatogram (TIC) of 10 fmol acetylated BSA in 500 fmol six-protein mix.
(a) Information dependent acquisition (TIC of EMS).  (b) Precursor ion scanning at m/z 126.1 (TIC of precursors).  (c) MIDAS (TIC of MRM).17

 
 

Acetylated BSA – Mascot Result

A database (Mascot) search on the MIDAS acquisition of 5 fmol acetylated BSA in an excess of a 6 protein mixture digestion has led to the identification of a number of lysisne acetylated peptides and none of the non-modified matrix in which these peptides were spiked (Figure 23). 
This demonstrates the power of selectivity that MIDAS has.

 
Figure 23:  Mascot result of 5 fmol ACBSA into 500 fmol 6 protein mix (93 MRMs).
 
 

As can be seen from the composite product ion spectrum of one of the lysine acetylated peptides, great y ion coverage facilitates confirmation of the sequence and the position of lysine acetylation as K8 (Figure 24).

 

Figure 24:  Composite product ion spectrum and Mascot result of a lysine acetylated peptide.

 
 

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Ubiquitin (Ub) is a small regulatory protein that is found in almost all tissues of eukaryotic organisms.  Ubiquitination (also known as ubiquitylation) is an enzymatic, post-translational modification (PTM) process in which an ubiquitin protein is attached to a substrate protein.  Small Ubiquitin-like Modifier (SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a PTM involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle.16 

Attachment to lysine is through a glycine/cysteine (GC) c-terminus on both Ub and SUMO.8,9 

Both modes are important and occur via an enzyme cascade (Figure 25).

 

Figure 25:  Ubiquitination enzyme cascade. 

 
 

Tryptic ubiquitin isopeptides

After activation ubiquitin attaches to acceptor lysine forming an isopeptide bond (Figure 26).  Trypsin yields a GC tag but no diagnostic ions are formed.  The mass addition of 114.0429 must be relied upon for identification.  Our method exploits the fact that isopeptides have an additional N-terminus (cf. linear peptides).

 

Figure 26:  Activated ubiquitin attaching to an acceptor lysine to form an isopeptide bond.

 
 

The peptide can be derivatised at the N-termini using reductive methylation (dimethyl labeling, DML).  It has been well reported that this generates a strong a1 ion upon CID (Figure 27).10  It has also been postulated that isopeptides will have an extra a1 ion at m/z 62 and a b2 ion at m/z 147.  Deuterated reagents can be used to generate a much more selective m/z 147 ion (cf. the m/z 143 ion if non-deuterated reagents are used).

 

Figure 27:  N-termini peptide derivatisation using reductive methylation and the resulting CID spectra showing the fragment ions.

 
 

Ubiquinated ubiquitin on lysine 48 (Figure 28) shows a strong ion at m/z 62 and an additional ion at m/z 147.  The presence of the a1 ion at m/z 118 confirms that it is an isopeptide.

 

Figure 28:  MS/MS spectrum of Ub isopeptide (DML).

 
 

Figure 29 shows the MS/MS spectrum of the low mass region with an isotag.  A large m/z 62 ion and smaller, but highly selective m/z 147 is also produced.  Taken together with the backbone a1 ion and increased b ion coverage makes identification of the modified peptide easier.

 

Figure 29:  MS/MS spectrum of the low mass region with a labeled isotag.

 
 

Figure 30 shows the same peptide but without the GC tag i.e. not ubiquitinated. 
The a1 backbone ion is still generated as expected (DML has worked) but no a1’ or b2’ ions are present confirming that this is not an isopeptide.

 
Figure 30:  MS/MS spectrum of low mass region without isotag.
 
 

The digest used in figure 31 was DML labeled and analysed by IDA on qStar. 
The presence of a1’ and b2’ ions can be clearly seen along with extensive b ions on the backbone resulting in facile identification of the peptide.

 

Figure 31:  MS/MS spectrum of 1D gel digest from Ub pulldown material.

 
 

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Unlike Ub, SUMO generates a large tag upon tryptic digestion.  However, we have found that trypsin (or something in the trypsin) cleaves the c-terminal to glutamine (Q) leaving MS useful tags.  This has been found by searching the data with variable modes on K of G, GG, TGG, QTGG, QQTG, GQQQTGG (Figure 32).
The use of DML may improve detection.

 

Figure 32:  Consecutive residue addition to lysine – CRA(K).

 
 

Tags are indeed found and they generate diagnostic ions such as b3’ for TGG tag (Figure 33) and b2’ and b4’ ions for GGTQ tag (Figure 34).

 

Figure 33:  SUMO TGG peptide from ‘trypsin’ digestion.

 

Figure 34:  SUMO QTGG peptide from ‘trypsin’ digestion.

 
 

The product ion spectrum of the DML isopeptide carrying the QTGG isopeptide contains the predicted a1’ b2’ and b4’ diagnostic ions in addition to a multitude of backbone sequence ions (Figure 35, left).  Taken together, these ion series facilitate the confident assignment of this spectrum to the backbone sequence, the QTGG isotag, and the site of modification as K4.

The product ion spectrum of the DML isopeptide carrying the TGG isopeptide contains the predicteda1’ b2’ and b3’ diagnostic ions in addition to a multitude of backbone sequence ions (Figure 35, right).  Taken together, these ion series facilitate the confident assignment of this spectrum to the backbone sequence, the TGG isotag and the site of modification as K4.

 
Figure 35:  Tandem MS/MS spectrum showing effect of DML on CRA(K) peptides.12
 
 

Data Independent Acquisition (DIA)

A mixture of DML E.coli tryptic digest and DML synthetic isopeptides were mixed and run using the Data Independent Acquisition (DIA) approach termed SWATH (Figure 36).  For complex mixture analysis the use of Data Independent Acquisition (DIA) scanning routines negates the duty cycle limitations associated with classical Data Dependent Acquisition (DDA).  DDA analysis requires that the precursor of interest is selected for fragmentation.  In a DIA workflow, ALL ions are fragmented in a non-biased manner.  One method of DIA segments the duty cycle into a series of user-defined SWATH m/z windows.  

The 5600 acquires an MS scan followed by 80 5 Da SWATH MS/MS acquisitions over the m/z range 400-800 Th.  This results in every putative precursor being selected for MS/MS in a data independent manner, therefore, enabling a far more comprehensive analysis by which precursors of interest can be implicated by extraction of diagnostic ions.

A mixture of E. coli digest and Ub-isopeptides is dimethyl labelled and analysed by DIA on a 5600 (Figure 36). Post-acquisition ion extraction of the a1’ (m/z 62.09) and b2’ (m/z 147.11) ions is then utilised to screen for the modified peptides.

 

Figure 36:  Mixture of E. coli digest and Ub-isopeptides is dimethyl labelled and analysed by DIA on a 5600.

 
 

Figure 37 shows that in one SWATH window all precursors between 504.0 and 510.0 are fragmented.  XIC overlay of two diagnostic ions shows co-elution.  In practice this would have to be repeated for all 80 (5 Da) windows so a software script is required to facilitate this process. 

 

Figure 37:  XIC overlay of two diagnostic ions.

 
 

At the time of elution of the diagnostic ions the MS/MS spectrum can be extracted (Figure 38).  This may contain other precursor ions but clearly the main sequence ions enable easy identification.  If this is not the case, the MS scan for this time and for this SWATH window can be added and all the precursors compiled to give an inclusion list for subsequent DDA runs.

 

Figure 38: MS/MS spectrum at 27.47 minutes.

 
 

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The future direction of PTM analyses will be to incorporate the analysis of many different PTMs into a single workflow that will require appropriate serial enrichments coupled with specific chemistries, LCMS/MS approaches, and informatics capable of handling such complex data.  As in many other areas of Proteomics, the future will surely demand these analyses to be performed in a quantitative manner.

Our approach of developing MEDUSA (Mass spectral Enhanced Detection of Ubls with SWATH Acquisition) is a step towards the ultimate goal.

  • MEDUSATM – DIA + SWATH + mTRAQ
  • SEPTM – Serial enrichments of PTMs
  • ETD etc. retain modification
  • Combinatorial detection with quantification
  • Other PTM targets
 
 

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The analysis of PTMs by LCMS/MS is a growth area with massive potential to deliver in many areas of biomedical sciences.  These analyses are non-trivial and require the integration of biochemical, LC, MS, and informatics workflows to fully exploit their power.  The field is very much in its infancy with rapid developments in every aspect of these workflows required to enable delivery of their full potential.

 
 

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  1. Wilkins, Marc (Dec. 2009). "Proteomics data mining". Expert review of proteomics (England) 6 (6): 599–603.

  2. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humphery-Smith I. (1995). "Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium". Electrophoresis 7 (7): 1090–94.

  3. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humphery-Smith I. (1995). "Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium". Electrophoresis 7 (7): 1090–94.

  4. Wilkins, MR, Williams, KL, Appel, RD, Hochstrasser, DF, ed. (1997). Proteome Research: New Frontiers in Functional Genomics. Springer-Verlag.

  5. Stojanov & McDermott   Expert Reviews in Molecular Medicine 2005 Oct 7;22 1-18

  6. Yang XJ, Seto E (2008) Lysine acetylation: codified crosstalk with other posttranslational modifications. Mol Cell 31: 449–461.

  7. Huang JY, Hirschey MD, Shimazu T, Ho L, Verdin E (2010) Mitochondrial sirtuins. Biochim Biophys Acta 1804: 1645–1651.

  8. Komander D. 2009. The emerging complexity of protein ubiquitination. Biochem Soc Trans 37:937-953.

  9. Hay RT. 2005. SUMO: a history of modification. Mol Cell 18:1-12.

  10. Chicooree et al., 2013. J Am Soc Mass Spectrom. 24:421-430.

  11. Chicooree et al., 2013. Rapid Commun Mass Spectrom. 27:127-134.

  12. Chicooree et al., 2013. Rapid Commun Mass Spectrom, DOI: 10.1002/rcm.6670.

  13. Cohen, P. (2001) "The role of protein phosphorylation in human health and disease. The Sir Hans Krebs Medal Lecture." Eur J Biochem 268:5001-10.

  14. Wang, B., Malik, R., Nigg, E.A., and Körner, R.; Anal. Chem. 2008, 80, 9526-9533.

  15. Taouatas, N., Drugan, M.M., Heck, A.J., Mohammed, S.; Nat. Methods 2008 May;5(5):405-7.

  16. Hay RT (Apr 2005). "SUMO: a history of modification". Mol. Cell 18 (1): 1–12.

  17. Griffiths JR, et al. J Am Soc Mass Spectrom. 2007 Aug;18(8):1423-8. Epub 2007 May 6.
 
 

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In this session, Dr Duncan Smith and Dr John Griffiths (MS-Insight Ltd and Manchester University) explain the principles behind the analysis of a number of key post-translational modifications (PTMs) using LC-MS/MS techniques. They will begin with a brief overview of bottom-up proteomics, explaining how proteins are converted to peptides, which are then analysed using electrospray LC-MS/MS. They will move on to talk about the various MS scanning techniques, which may be used for PTM analysis. Next, they will discuss in turn, how phosphorylation, acetylation, ubiquitination and SUMOylation may be analysed using mass spectrometry, drawing upon examples from their own research at the University of Manchester. Finally, they will give their own opinions on the ways in which they see the future of PTM analysis heading.

Dr Peter Tranchida

Dr John R Griffiths
Director
MS-Insight Ltd.
UK

Josep Miquel Serret

Dr Duncan L Smith
Director
MS-Insight Ltd.
UK

Key Learning Objectives:

  • Understand the general principles of bottom-up proteomics
  • Description of some of the major MS scanning techniques for the detection of PTMs
  • Description of phosphoproteomics including enrichment and analysis
  • How to apply MIDAS to targeted analysis of phosphorylation and acetylation
  • Application of novel strategies to ubiquitination and SUMOylation determination
  • What the future holds for PTM analysis