LC-MS vs ELISA / Flow Cytometry / FRET / ELISA / Sandwich ELISA / SIMOA

• The European Medicines Agency (EMA) similarly states, “A SNR between 3 or 2:1 is generally considered acceptable for estimating detection limit”
• a recommended guideline such as SNR < 10:1 for the standard or sample

Products

• PTMScan
  • Protein 에 antibody 붙인 후 LC-MS 함.
  • Advantage
    • ◆ 적은 양의 protein 도 identification 가능함

LC-MS

	Enriched by	Separated by		Detected by
Traditional (gas?) LC		affinity to the packing materials and the solvent		a UV detector and measures the intensity of absorbance.
HPLC			Faster separation than traditional LC (gas?)	the number of ions
MS		mass
LC-MS (ie HPLC+MS) For sure, LC-MS doesn't involve the use of an antibod y.		Mass & Affinity	separated followed by ionization and separation of the ions on the basis of their mass/charge ratio. The separated ions are then directed to a photo or electron multiplier tube detector, which identifies and quantifies each ion. The ion source is an important component in any MS analysis, as this basically aids in efficient generation of ions for analysis. To ionize intact molecules, the ion source could be APCI (Atmospheric Pressure Chemical Ionization), ESI (Electrospray Ionization), etc. to name a few popular ones. The choice of ion source also depends on the chemical nature of the analyte of interest i.e. polar or non-polar.
PTMScan Hybrid LC-MS/MS	immunoaffinity capture using analyte-specific ligands or antibodies → denaturation, trypsin → release of signature peptide →	Mass & Affinity	separation of the ions on the basis of their mass/charge rati	Antibody the number of ions

LC-MS vs ELISA

LBA is a general term for analytic binding assays; in this case I am referring to antibody-based quantitative assays. This would include various forms of plate-based ELISAs, electrochemical (ECL) assays such as MSD, bead-based assays like Simoa etc.

Advantage of Mass spec	Disadvantage of mass speck
high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein	specialized personnel and expensive instrumentation.
	there are only few protein MS applications routinely used in clinical chemistry laboratories today. method development and method validation: time-consuming work
-	regulatory context: In vitro diagnostic (IVD) certification in Europe and FDA approval in the US is common practice for immunoassay kits and instruments. In the field of protein mass spectrometry, such certifications are certainly not common practice today. In fact, only very few instrument vendors propose such certificat[ions]; limited choice of instruments, mostly triple quadrupoles, and no protein assay is yet FDA or CE certified for diagnostic applications. This
-	sensitivity of MS instruments is below immunoassays,
-	-

Serum Protein Measurement Methods

ELISA / LBA	LC-MS/MS
Pros: High sensitivity High throughput Traditional methodology Minimal sample prep Cons: Assay specific reagents Long lead times Poor standardization Specificity concerns Difficult to multiplex	Pros: Highly selective Faster assay development Ability to multiplex Can be combined with enrichment Cons: Expensive instrumentation Extensive sample prep Requires an internal standard

Flow cytometry

Like a microscope
Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations, and analyzing cell size and volume.
What is flow cytometry antibody? Flow cytometry directly detects antigens using fluorescent conjugated antibodies, each corresponding to a different protein in
-	Limitations to flow cytometry include the facts that the laser can only analyze one cell at a time, cells must be in suspension to be analyzed (thereby restricting the analysis of tissue), highly trained operators are required, and cells must be viable to be analyzed.
-
-

FRET (Fluorescence Resonance Energy Transfer) ASSAY

• a distance-dependent interaction between two fluorophore
• During FRET a donor fluorophore becomes excited by a light source, however it does not emit light following excitation but instead transfers its energy to an acceptor fluorophore. The acceptor fluorophore absorbs this and then produces a detectable light emission
• Protein 간의 interaction을 볼 수 있음.
  • ligand binding to a receptor, has taken place.
  • Phosphorylation 되었는지 여부

R-FRET

• (time-resolved fluorescence resonance energy transfer)
• Purpose
  • To analyze binding event, binding affinity of two binding partners, high-thoroughput drug screening, PPI ,
  • Js: What abount quantification of (already-bound) protein complex?
• 320-340 nm로 빛을 excitation하면 LANCE Europium chelate가 labeling된 donor fluorophore에서 에너지가 FRET (~ 10 nm) 되어 acceptor fluorophore인 ULight™ dye를 반응 시킨 후 665 nm에서 빛이 emission 됩니다. 양 쪽 dye 간의 거리가 10 nm 이상일 경우에는 ULight™ dye로 에너지 전달이 안되기 때문에, LANCE Europium dye의 615 nm emission 결과값만 얻을 수 있습니다.
• Application
  • Cell lysate: O
  • Js: this is not applicable to in vivo? Hioki Takeshi: brain lysate can be used.
  • Biofluid 도 가능하겠지 . to be confirmed

ELISA

• a plate-based assay technology designed for use in detecting and quantifying substances such as peptides, proteins, antibodies, and hormones.
• ELISA technology is also sometimes referred to by other names such as “enzyme immunoassay (EIA).”
• In an ELISA, an antigen immobilized on a solid surface must be mixed with an antibody bound to an enzyme. To achieve detection, bound enzyme activity is assessed by incubation of a substrate that yields a measurable product.

Sandwich ELISA

		principle	example
labeling material (conjugated to detection antibody)	MSD: Sulfo tag	- SULFO-TAG is a small hydrophilic molecule (approximately 1 kDa) - Easily conjugated to any detection antibody - Generates light (emission at 620 nm) when Electricity is applied to the bottom plate → quantitative measure of analytes	-Capture & detection antibody detect different and non-overlapping region or epitopes of the antigen. -After immobilization by a capture antibody , a detection antibody is added,
Detection antibody			anti pS65-Ub
Analyte			pS65-Ub
Capture antibody		Plate에 붙여놓고 전기 흘러야 하니까 이게 필요한 듯.	Biotinylated TUBE2
쪽 MSD plate coated with Streptavidin		여기에 전기 흘림

• Assay 순서:

  • the well of an ELISA plate to be coated with a capture antibody.

• → The analyte or sample is then added,

• → followed by a detection antibody.

• Advantage

  • a sandwich ELISA is 2-5 times more sensitive than direct or indirect ELISAs.
  • Sandwich ELISA also delivers high specificity as two antibodies are used to detect the antigen. (so the antigen does not need to be purified prior to the assay)

• Disadvantage

  • if a standardized ELISA kit or tested antibody pair is not available, antibody optimization has to be worked out. But Antibody optimization can be difficult because cross-reactivity may occur between the capture and detection antibodies

SIMOA (Single Molecule Array)

‘digital ELISA’ , immunoassay, Ab is used

Uncertain Spans

location	transcription	uncertainty
LC-MS table column header gap	the gap between Detected by block and right-most empty column	The right-most column appears to repeat Antibody / the number of ions for the Hybrid LC-MS/MS row; column boundaries with the inner cells are visually ambiguous.
LC-MS row	Traditional (gas?) LC literal text	The (gas?) parenthetical is the source’s own self-question, transcribed as written; the ? belongs to the source.
FRET section	What abount quantification of (already-bound) protein complex?	abount reads literally as a typo of about in the source; transcribed as written.
Sandwich ELISA principle row	quantitative measure of analytes ending arrow	The arrow direction at the end of the principle bullet is partly cut by the cell border.