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  • Intact protein molecular weight using MS by MALDI (top) or ESI (bottom).
  • MALDI-MS primarily results in singly charged molecules, while ESI-MS can give multiply charged species (especially for proteins).

mwd1

mwd2 
Quantitative experiments can provide vital information regarding changes in biological systems that occur as a function of disease, drug treatment,
fig3
 exposure to a chemical toxicant, or some other perturbation. Technical capabilities for quantification include the classical selected reaction monitoring (SRM, Figure 3) experiment with isotopically labeled internal standards (6500 Q-Trap, Figure 7), metabolic labelling (SILAC), area-under-the-curve-based label-free relative quantification (Orbitrap Fusion, 5600 TTOF, Figure 4) and SWATH (Figure 6, (5600 TTOF)). In addition, isobaric labeling strategies using tandem mass tags can also be performed (Figure 5). 

 

SWATH (sequential window acquisition of all theoretical fragment ion spectra) MS is a data independent acquisition (DIA) method which aims to complement tradition mass spectrometry based proteomics techniques. It allows a complete and permanent recording of all fragment ions of the detectable peptide precursors present in a biological sample. 
fig6-swath

An example of SWATH experiment. First, a data dependent acquisition run was used to build a protein library for all samples in the project. Then data independent acquisition runs for each sample were used for comparison.  (A) Protein list of the sample; (B) peptide list from protein selected from A ; (C) fragment ions from one peptide selected from B; (D) fragment ions march in the library.

An example of SWATH experiment. First, a data dependent acquisition run was used to build a protein library for all samples in the project. Then data independent acquisition runs for each sample were used for comparison.  (A) Protein list of the sample; (B) peptide list from protein selected from A ; (C) fragment ions from one peptide selected from B; (D) fragment ions march in the library.
srm_figureSelected reaction monitoring (SRM) is a targeted mass spectrometry technique that is emerging in the field of proteomics as a complement to untargeted shotgun methods. SRM is particularly useful when predetermined sets of proteins, such as those constituting cellular networks or sets of candidate biomarkers, need to be measured across multiple samples in a consistent, reproducible and quantitatively precise manner. 
 

 

 
SILAC
Stable isotopic labeling with amino acid in cell culture(SILAC) is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometry based quantitative proteomics.

iTRAQ
iTRAQ protein quantification technique suite for unbiased untargetedfig5 biomarker discovery. It is ideally applied for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and provides relative quantification. It allows to compare up to 8 samples at one time. (See workflow on the right)

TMT
The Tandem Mass Tag Reagents (TMT) are designed to enable identification and quantitation of proteins in different samples using tandem mass spectrometry. The TMT 10plex label reagents share an identical structure with TMTzero, TMTduplex, and TMTsixplex reagents but contain different numbers and combinations of 13C and 15N isotopes in the mass reporter.
 
Post-translational modifications (PTMs) of protein plays a crucial role in generating the heterogeneity in proteins and also help in utilizing identical proteins for different cellular functions in different cell types. We offer PTM mapping service for many different PTMs. Left two HCD MS/MS spectra for a nitrotyrosine-containing peptide (A) and a phosphopeptide (B). In both instances, the site of modification is confirmed by the presence of b and y ions flanking the site of modification.
Left two HCD MS/MS spectra for a nitrotyrosine-containing peptide (A) and a phosphopeptide (B). In both instances, the site of modification is confirmed by the presence of b and y ions flanking the site of modification.

Post-translational modifications (PTMs) of protein plays a crucial role in generating the heterogeneity in proteins and also help in utilizing identical proteins for different cellular functions in different cell types. We offer PTM mapping service for many different PTMs. 

ptmLeft two HCD MS/MS spectra for a nitrotyrosine-containing peptide (A) and a phosphopeptide (B). In both instances, the site of modification is confirmed by the presence of b and y ions flanking the site of modification.

 
Label-free quantification is a mass spectrometry method which aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantification, this technique does not use a stable isotope containing or other labeling compound.

A scheme is shown on the right.  LC-MS/MS data files representing sampleslabel_free from multiple experimental conditions are aligned by accurate mass and retention time, and MS/MS spectra are used to assign peptides to “features” within the alignment. Peptide-level intensities are rolled up to the protein level to calculate estimated protein-level fold changes. 
SWATH (sequential window acquisition of all theoretical fragment ion spectra) MS is a data independent acquisition (DIA) method which aims to complement tradition mass spectrometry based proteomics techniques. It allows a complete and permanent recording of all fragment ions of the detectable peptide precursors present in a biological sample. 
 
swath1An example of SWATH experiment. First, a data dependent acquisition run was used to build a protein library for all samples in the project. Then data independent acquisition runs for each sample were used for comparison.  (A) Protein list of the sample; (B) peptide list from protein selected from A ; (C) fragment ions from one peptide selected from B; (D) fragment ions march in the library.
 
Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that is emerging in the field of proteomics as a complement to untargeted shotgun methods. SRM is particularly useful when predetermined sets of proteins, such as those constituting cellular networks or sets of candidate biomarkers, need to be measured across multiple samples in a consistent, reproducible and quantitatively precise manner.
srm_figure
SRM experiment: (A)60 transitions were monitored for 10 peptides (from 6 proteins) heavy (synthesized) and light (samples) version. The rations of H:L were then used to compare among 48 biological samples. Heavy and light peptide intensity comparison at the retention time 22.8 (C) and 26.7 (B) min.
 
AAA
Simultaneous analysis of 45 amino acids in physiological fluid (urine, serum, plasma) with LC/MS/MS (6500 Qtrap). The isomeric and isobaric amino acids are separated using LC and unique chemistries. Internal standard for each analyte, leading to accurate and
precise analyte identification and quantitation. Small sample requirement (40uL). Wide dynamic range, with LLOQ and ULOQ of <1 uM to
>10,000 uM, respectively
 
The formation of N- and C- terminally truncated proteins by proteolytic processing represents an important irreversible post-translational modification. These newly generated protein variants can exhibit different biological functions compared to their genetically encoded substrates. We offer optimized methods to identify those novel-formed N- and C-terminis. (Sample requirement: good in quantity and quality.)
 

ncterminalProtein/Peptide Dimethyl labeling

  • Addition of dimethyl by reductive amidation
  • Reacts with N-terminal and lysine residues

Reaction Condition

  • Optimized labeling reaction pH = 5.0
  • 4% formaldehyde
  • Na cyanoborohydride
  • Incubate 5 mins at RT

Data Analysis

  • Dimethylated lysine should be included in database search
  • Result needs to be manfully evaluated
The  premise of this method is that when proteins are enzymatically digested in the presence of O18 water,  all specifically cleaved internal peptides willlysine exchange  the O18 tag except for the peptide originating from the carboxyl-terminus of the protein.
ctermExample: β-lactoglobulin was digested with trypsin in either 16O and 16O/18O labelled water. Internal peptides were find both 16O and 18O species, but C terminal peptide will not exchange with 18O H2O