Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives

Table 1

Overview of the SCX chromatography methods used for PTM analysis described in this paper.

Sample type	Protein amount/cell number	A number of fractions collected	Mass spectrometer used	Experimental workflow	Other details	PTM analysis	References
HeLa cells	8 mg protein	4 early fractions from each gel band	LCQ DECA XP ion trap	SDS-PAGE (10 gel bands) → SCX → LC-MS/MS		2002 phosphorylation sites identified from 967 proteins	[6]
HeLa cells	0.5 mg protein	10 fractions	LTQ-Orbitrap	SCX → TiO2 → LC-MS/MS		722 nonredundant phosphorylation sites	[7]
Human Chang liver cells	1.5 mg protein	40 fractions	Q-TOF and LCQ	SCX → TiO2 → LC-MS/MS		1035 phosphorylation sites from 607 phosphoproteins	[8]
Escherichia coli cells	20 mg protein	10 fractions	LTQ-Orbitrap	SCX → TiO2 → LC-MS/MS	Lys-C digestion followed by trypsin digestion	81 phosphorylation sites from 79 proteins	[9]
Bacillus subtilis cells	10 mg protein	15 fractions	LTQ-Orbitrap or LTQ-FT	SCX → TiO2 → LC-MS/MS	Lys-C digestion followed by trypsin digestion	78 phosphorylation sites from 78 proteins	[10]
HeLa cells	40 mg protein	25 fractions	QSTAR ELITE	IMAC → SCX → LC-MS/MS		~4512 phosphorylated sites	[11]
HEK293 cells	1 mg protein	40 fractions	LTQ-Orbitrap	SCX → LC-MS/MS	Lys-N, Lys-C, and trypsin digestion	5036 nonredundant phosphorylation sites	[4]
Human embryonic stem cells	10 mg protein	12 fractions	LTQ-Orbitrap	SCX → IMAC → LC-MS/MS	CID and ETD fragmentation methods used in mass spectrometry	10844 nonredundant phosphorylation sites	[12]
Membrane fraction of human teratocarcinoma Nt2/d1 cells	5 × 10^6 cells	~30 fractions	LTQ-Orbitrap	SCX → LC-MS/MS	Lys-C digestion followed by trypsin digestion	116 N-acetylated sites	[13]
HEK293 cells	12.3 mg protein/6 × 10^7 cells	49 fractions	LTQ XL Linear ion trap	SCX → LC-MS/MS	CID and ETD fragmentation methods used in mass spectrometry, Lys-N digestion	N-acetylated peptides	[14]
Cytoplasmic, nuclear, and membrane fractions from Kc 167 cells (Drosophila melanogaster)	n/a	n/a	HCT ion trap, LTQ linear ion trap, and XCT-Ultra ion trap	SCX → COFRADIC → LC-MS/MS		861 N-acetylated sites	[15]
HEK293 cells	1 mg protein	~40 fractions	LTQ-Orbitrap	SCX → LC-MS/MS	Lys-N, Lys-C, and trypsin digestion	1391 N-acetylated peptides	[16]

SCX: strong cation exchange; PTMs: post-translational modifications; IMAC: immobilized metal affinity chromatography; ETD: electron transfer dissociation; CID: collision-induced dissociation; COFRADIC: combined fractional diagonal chromatography; LC-MS/MS: liquid chromatography-tandem mass spectrometry; n/a: data not available.