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PTMScan® Discovery Proteomic Services

PTMScan Discovery Proteomic Services Workflow

PTMScan® Discovery allows discovery of hundreds to thousands of novel sites of post-translational modification (PTM) in a single LC-MS/MS run.

PTMScan Discovery features

  • Antibody enrichment of PTM-containing peptides using PTM and motif antibodies developed by CST
  • LC-MS/MS for quantitative analysis of enriched peptides
  • Can be used on samples from many different species including, but not limited to, human, mouse, rat, Drosophila, and Arabidopsis

If you prefer to perform your own peptide enrichment and LC-MS/MS, see the list of our PTMScan Antibody Kits.

Perform follow-up on many candidates identified in PTMScan Discovery by using modification state-specific and total protein antibodies from CST, which are produced and validated in-house for multiple applications.

Protein ubiquitination is involved in many cellular processes including proteasomal degradation, endocytosis, DNA repair, cell cycle regulation, and gene expression. Abnormal ubiquitination is involved in diseases such as cancer, neurodegeneration, and metabolic syndrome.

UbiScan® technology for ubiquitination proteomics employs a proprietary antibody against the di-glycine (K-ε-GG) remnant that is left on ubiquitinated lysine residues after trypsin digestion. This ubiquitin remnant motif antibody is used to enrich ubiquitinated peptides from trypsin digested samples prior to LC-MS/MS analysis for quantitative profiling of thousands of non-redundant ubiquitinated sequences. (Other ubiquitin-like modifiers also leave K-ε-GG remnants, for example NEDD8 and ISG15.)

UbiScan Process

View webinar to see an example of how PTMScan technology using the K-ε-GG remnant motif antibody can be applied in large-scale quantitative analysis of the ubiquitylome.

UbiScan Proteomics Service

Target Description Motif Reference Data
Target Description Motif Reference Data
Ubiquitin Remnant K-ε-GG Mouse Liver | XLS | RAW

Small Ubiquitin-like Modifier (SUMO) proteins are small proteins that reversibly modify target proteins through covalent attachment at lysine residues. Protein SUMOylation plays a critical role in a number of cellular processes such as:

  • Nuclear transport
  • DNA replication and repair
  • Mitosis
  • Signal transduction

PTMScan® Sumoylation Remnant Motif technology for SUMOylation proteomics employs a proprietary antibody to enrich SUMOylated peptides from WaLP digested samples. WaLP, a unique protease with specificity for threonine, valine, alanine, and serine, cleaves at aliphatic residues on the SUMO C-terminus to yield a di-glycine (K-ε-GG) remnant that is tagged onto lysine residues of the SUMOylated substrate. The resulting K-ε-GG-containing peptides can then be identified using methods already developed for ubiquitin-profiling (UbiScan® technology) (Figure 1). Thus, the same sample can be subjected to both SUMO and ubiquitin profiling by digesting the sample with either WaLP or trypsin, respectively.

Specific SUMO-proteases (SENP 1 and 2) were used to validate that the K-ε-GG sites identified by the PTMScan® Sumoylation Remnant Motif technology result from SUMOylation and not from ubiquitination (Figure 2). This ensures ubiquitinated peptides do not contaminate SUMOylated samples.

PTMScan® Sumoylation Remnant Motif Schematic Representation

A schematic representation of the strategy used to enrich SUMOylated (left) versus ubiquitinated (right) peptides using the K-ε-GG remnant motif antibody.

PTMScan® Sumoylation Remnant Motif K-ε-GG peptides

Bar graphs of results from quantification of K-ε-GG peptides derived from SUMOylated (A) and ubiquitinated (B) proteins on treatment with SENP 1 and 2. SUMOylated peptides decrease with treatment while ubiquitinated peptides are unchanged, consistent with western blotting data.

MethylScan Process

Representation of different methylation antibodies and their target modifications.

Protein methylation is a common post-translational modification (PTM) that mostly occurs on arginine and lysine residues. Arginine methylation regulates processes such as RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription.

MethylScan® technology for methylation proteomics uses proprietary methyl-arginine (Me-R) or methyl-lysine (Me-K) antibodies to enrich methyl-containing peptides from protease digested samples. Methylation antibodies from CST are rigorously tested, including in peptide blocking experiments and peptide arrays to confirm specificity and sensitivity. Each of these reagents is each optimally designed and formulated to recognize only its respective form of methylated arginine and lysine residue.

View a publication demonstrating MethylScan® methylation proteomics.

MethylScan Dot Matrix

Quantitative analysis of arginine monomethylation in mouse brain and embryo: Each dot in the scatter plot represents a unique arginine monomethylated peptide identified using PTMScan® Mono-Methyl Arginine Motif [mme-RG] Kit #12235. The x-axis is the log10 value of the total intensity of the representative peptide for a methylation site in mouse brain and embryo, and the y-axis shows the log2 ratio of intensity of the peptide in mouse brain vs. embryo. A cutoff of 5-fold was set to indicate increased arginine monomethylation peptide abundance in either brain (green dots) or embryo (red dots). For the methyl peptides that uniquely existed in a specific tissue, arbitrary log2 ratios of 15 (brain specific) and -15 (embryo specific) were assigned. Several representative enriched brain and embryo proteins are highlighted on the graph.

MethylScan® Proteomics Services

Target Description Motif Reference Data
Target Description Motif Reference Data
Mono-Methyl Arginine R-Me Mouse Embryo | XLS | RAW
Asymmetric Di-Methyl Arginine R-2Me(a)  
Symmetric Di-Methyl Arginine R-2Me(s)  
Pan-Methyl Lysine K-Me, K-2Me, K-3Me  
Acylscan Workflow

Profiling lysine acylation in liver peptides from wild type and Sirt5 knockout mice: Venn diagram showing the degree of overlap of sites identified using the four indicated acylation-specific antibodies. See full dataset here.

Lysine residues are subject to a wide range of post-translational modifications due to the positively charged ε-amino group side chain. Acyl group transfer from the metabolic intermediates acetyl-, succinyl-, malonyl-, glutaryl-, butyryl-, propionyl-, and crotonyl-CoA all neutralize the positive charge of lysine and confer structural alterations affecting substrate protein function. Cellular functions regulated by acylation include cell cycle regulation, mitochondrial metabolism, cytoskeletal regulation, protein-protein interactions and others.

Acylscan Venn Diagram

AcylScan™ technology for acylation proteomics uses proprietary acetyl- (Ac-K), glutaryl- (Glut-K), malonyl- (Mal-K), propionyl- (Prop-K), and succinyl-lysine (Succ-K) antibodies to enrich their respective acyl-containing peptides from trypsin digested samples prior to LC-MS/MS analysis.

AcylScan™ Services

Service Target Description Motif Reference Data
Service Target Description Motif Reference Data
AcetylScan® Acetyl-Lysine Ac-K Mouse Liver | XLS | RAW
GlutarylScan™ Glutaryl-Lysine Glut-K  
MalonylScan™ Malonyl-Lysine Mal-K  
PropionylScan™ Propionyl-Lysine Prop-K  
SuccinylScan™ Succinyl-Lysine Succ-K  

PTMScan Discovery Workflow

The reversible addition of phosphate groups by protein kinases can activate or inhibit protein function. Phosphorylation signaling cascades, for example the MAP kinase signaling pathway, are important for transmitting information from outside to inside the cell where it ultimately results in modulation of gene transcription. Aberrant phosphorylation is widespread in diseases including cancer, diabetes, and neurodegeneration. Therefore, identifying the phosphorylation status of proteins is crucial to understanding cellular processes in both health and disease.

Apply PhosphoScan® proteomics for

  • Pathway profiling
  • Biomarker discovery
  • Target validation

Why consider antibody enrichment for phosphorylation proteomics?

  • More focused discovery of phosphosites based on amino acid (S/T/Y) or motif of interest than IMAC.
  • PTMScan® methods and IMAC enrich different pools of phosphopeptides making them highly complementary. For the most complete dataset customers should use both types of enrichment. View data.

PTMScan Discovery Dot Matrix

Profiling tyrosine phosphorylation in MKN-45 cells. Peptides from MKN-45 cells treated for 2hr with DMSO (control), 1 μM SU11274 ,or 200 nM Staurosporine #9953 for were enriched using the PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit #8803 (above). In parallel, total protein levels were profiled by running unenriched material in LC-MS/MS (below). Peptides that decreased in abundance with SU11274 or Staurosporine are indicated in red, peptides that increased with treatment in green. A % CV histogram of analytical replicates for each enrichment is shown on the right with the median % CV indicated in blue.

PhosphoScan Services

Note: Custom mixes of Ser/Thr motif antibodies can be made for services. Please inquire.

Target Description Motif Reference Data
Target Description Motif Reference Data
14-3-3 Binding Motif (R/K)XX(s/t)XP  
Akt Substrate RXX(s/t) Mouse Liver | XLS | RAW
Akt Substrate RXRXX(s/t) Mouse Liver | XLS | RAW
AMPK Substrate LXRXX(s/t) Mouse Liver | XLS | RAW
ATM/ATR Substrate (s/t)Q Mouse Liver | XLS | RAW
ATM/ATR Substrate (s/t)QG Mouse Liver | XLS | RAW
CDK Substrate (K/R)(s/t)PX(K/R)  
CK2 Substrate (s/t)(D/E)X(D/E)  
MAPK/CDK Substrate PX(s/t)P, (s/t)PX(K/R) Mouse Liver | XLS | RAW
PDK1 Docking Motif (F/K)XX(F/Y)(s/t)F/Y) Mouse Liver | XLS | RAW
PKA Substrate (K/R)(K/R)X(s/t) Mouse Liver | XLS | RAW
PKC Substrate (K/R)X(s/t)X(K/R) Mouse Liver | XLS | RAW
PKD Substrate LXRXX(s/t)  
PLK Binding Motif S(s/t)P Mouse Liver | XLS | RAW
tP Motif (s/t)P, (s/t)PP  
tPE Motif (s/t)PE Mouse Liver | XLS | RAW
tXR/tPR Motif (s/t)XR, (S/t)PR Mouse Liver | XLS | RAW
Phospho-Tyrosine (pY-1000) y Mouse Brain | XLS | RAW

The intrinsic and extrinsic apoptosis pathways involve a cascade of caspases. The human proteome contains thousands of known or putative caspase cleavage sites. The majority of caspase substrates are cleaved at an aspartic acid residue, generating fragments containing a carboxyl-terminal aspartate with a general DEXD motif with some variation.

PTMScan® technology for caspase cleavage proteomics uses a proprietary antibody to the DEXD motif to enrich caspase cleaved substrate peptides from trypsin digested samples prior to LC-MS/MS.

Caspase Sequencing

Caspase Cleaved Substrate Proteomics: The motif logo was generated from a PTMScan® LC-MS/MS experiment using 1044 nonredundant tryptic peptides with carboxy-terminal aspartates derived from HeLa cells treated with Staurosporine #9953 (1 μM, 3 hr) to induce apoptosis. Peptides were enriched using the PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Kit #12810. The logo represents the relative frequency of amino acids in each position leading up to the carboxy-terminal aspartate.

Cleaved Caspase Substrate Motif [DE(T/S/A)D] MultiMab Western Blot

Cleaved Caspase Substrate Motif [DE(T/S/A)D] MultiMab® Rabbit mAb mix #8698: Western blot analysis of NIH/3T3 and HeLa Cells, untreated (-) or treated with Staurosporine #9953 (1 μM, 3 hr; +), using Cleaved Caspase Substrate Motif [DE(T/S/A)D] MultiMab® Rabbit mAb mix (upper) or GAPHD (D16H11) XP® Rabbit mAb #5174 as a loading control (lower).

Caspase Cleaved Substrate Proteomics Service

Target Description Motif Reference Data
Target Description Motif Reference Data
Cleaved Caspase Substrate DEXD  

Protein N-terminal acetylation is a common post-translational modification (PTM) that typically occurs on newly translated proteins, but can also occur independently of new protein synthesis. N-terminal acetylation is a conserved modification from bacteria to higher eukaryotes. A group of enzymes, the N-terminal acetyltransferases (NATs) mediate this modification, with some NAT family members modifying nascent polypeptides on the initiator methionine, while other NATs acetylate the N-terminal residue post-excision of the initiator methionine. N-terminal acetylation regulates such processes as protein localization, protein-protein interaction, and protein stability. NATs have also been implicated in cancer, neurodegenerative disease, and other human hereditary lethal conditions.

N-AcetylScan® technology for N-terminal acetylation proteomics uses proprietary N-terminal acetyl (N-AC) antibodies to enrich N-acetyl-containing peptides from protease digested samples. N-acetylation antibodies from CST are rigorously tested, including ELISA, western blot, and PTMScan® testing to confirm specificity and sensitivity. This reagent is optimally designed and formulated to recognize only N-terminally acetylated peptides. PTMScan® Discovery Motif Logo

The Motif Logo was generated from an N-Terminal AcetylScan® LC-MS/MS experiment using 839 nonredundant tryptic peptides derived from mouse liver, brain, and embryo tissues immunoprecipitated with PTMScan® N-Terminal Acetyl Motif Immunoaffinity Beads. The logo represents the relative frequency of amino acids in each position starting from the N-terminal residue of each peptide within this data set.

N-Terminal AcetylScan® Service

Target Description Motif Reference Data
Target Description Motif Reference Data
N-Terminal AcetylScan Acetyl-NH2-X Mouse Tissue | XLS | RAW

Glycosylation, the covalent addition carbohydrate groups to proteins, is a common post-translational modification that affects protein structure, stability, and signaling. Glycosylation is of particular importance on membrane and secreted proteins, including immunoglobulins. N-linked glycosylation involves addition of the glycan groups to asparagine residues on proteins, with these glycans often containing a terminal sialic acid group. After protease digestion, sialic acid-containing glycopeptides can be enriched using immobilized metal affinity chromatography (IMAC), in which the positively charged metal ion will interact with the negatively charged sialic acid group.

Traditionally, mass spectrometry-based proteomic analysis of glycosylation has been focused either on identification of the peptides that were glycosylated or on the structure of the glycan itself, but not resolution of both peptide sequence and glycan structure at the same time. PTMScan® Glycosylation profiling provides both the peptide sequence and the glycan structure at the same time. This allows identification and quantification of hundreds to thousands of glycosylated peptides with the structure of the glycan group included. Using PTMScan® Glycosylation analysis the landscape of glycosylation in serum or media samples can be profiled, and changes in that landscape quantified to find glycosylation events regulated by a particular treatment.

MS/MS spectrum of a glycopeptide identified using PTMScan Glycosylation profiling

MS/MS spectrum of a glycopeptide identified using PTMScan Glycosylation profiling. Both the peptide sequence and the structure of the glycan are determined from the same spectrum. Fragment ions corresponding to peptide backbone (b and y ions) and glycan groups (structures of individual sugar units) are indicated.

From Bench to Bedside

PTMScan Discovery Workflow, A Case Study

CST performed a global survey of tyrosine kinase activity in non-small cell lung cancer (NSCLC) to identify novel disease drivers (1). A phosphotyrosine antibody was used to enrich phosphorylated peptides from 41 NSCLC cell lines and 150 NSCLC tumors prior to LC-MS/MS analysis. The analysis identified 4551 phospho-tyrosine residues on more than 2700 proteins. The tyrosine kinase ALK (anaplastic lymphoma kinase) was among the top 10 candidates for follow up analysis.

EML4-ALK Fusion

Further investigation revealed fusion of the N-terminus of EML4 (echinoderm microtubule-associated protein-like 4) with the C-terminus of ALK in some NSCLC cell lines and tumors. 3-7% of NSCLC patients express the fusion protein in their tumors indicating that the it is highly oncogenic (1-4). Cancer cells expressing the EML4-ALK fusion protein are sensitive to the small molecule ALK inhibitor crizotinib, and in 2011 the FDA approved crizotinib for the treatment of ALK positive NSCLC (2).

CST developed a highly specific and sensitive antibody, ALK (D5F3®) XP® Rabbit mAb #3633, which detects full length ALK and the EML4-ALK fusion protein. The FDA approved an immunohistochemistry (IHC) companion diagnostic test, which uses the ALK D5F3 clone licensed from CST (3). This will help physicians determine which NSCLC patients may be effectively treated with crizotinib.

IHC Analysis of ALK Expression

IHC analysis of paraffin-embedded human lung carcinoma with high (upper) and low (lower) levels of ALK expression using ALK (D5F3®) XP® Rabbit mAb #3633.


  1. Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J, Ren JM, Yuan J, Bakalarski CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J, Comb MJ (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131(6), 1190–203.
  2. FDA approves Xalkori with companion diagnostic for a type of late-stage lung cancer
  3. Ventana receives FDA approval for the first fully automated IHC companion diagnostic to identify lung cancer patients eligible for XALKORI® (crizotinib)