Table 2.

Mass spectrometric multiple reaction monitoring detection of protein glycation, oxidation, nitration, crosslinks, and branched-chain amino acids.

Analyte group	Analyte	R t (min)	Parent ion (Da)	Ion (Da)	CE (eV)	Neutral fragment loss (es)	Internal standard and amount added
Glycation	FL	28.5	291.0	84.3	31	H2CO2, fructosylamine	[2H4]FL, 0.3 pmol
	CML	28.5	204.9	130.1	12	NH2CH2CO2H	[13C6]CML, 0.25 pmol
	MG-H1†	11.6 & 12.5	229.2	114.3	14	NH2CH(CO2H)CH2CH=CH2	[15N2]MG-H1, 1.25 pmol
	Glucosepane	16.5	429.2	382.1	38	C2H5O	[13C6]Glucosepane, 0.25 pmol
	Pyrraline	17.9	255.2	84.3	23	2-CHO-5-HOCH2-pyrrole, H2CO2	[13C6,15N2]Pyrraline, 1.00 pmol
Oxidative damage	Dityrosine	19.9	361.2	315.3	15	H2CO2	[2H6]DT, 0.25 pmol
	GSA	32.2	114.0	68.0	15	H2CO2	[2H3]AAA, 2.5 pmol
Nitration damage	3-NT	23.2	227.1	181.2	13	H2CO2	[2H3]3-NT, 0.25 pmol
TG crosslink	GEEK	9.3	276.1	147.1	12	NH2CH(CO2H)CH2CH=C=O	Nε-(γ-[13C5]glutamyl) lysine, 2.5 pmol
BCAA	Leu	27.6	132.3	86.2	10	H2CO2	[2H3]Leu, 250 pmol
	Ile	31.5	132.3	86.2	10	H2CO2	[13C6]Ile, 250 pmol
	Val	8.2	117.8	72.0	19	H2CO2	[2H8]Val, 250 pmol

^†For MG-H1, R_t values for the 2 epimers are given. LC-MS/MS was performed as described previously [25, 35] with chromatography using two Hypercarb™ (5 μm particle size, 0.2 × 50 mm and 0.2 × 250 mm) columns, column switching, and elution with 0.1% trifluoroacetic acid (TFA) in water and custom acetonitrile (MeCN) gradient. Different chromatography conditions were used for assay of GEEK: the column was Hypercarb™ (2 μm particle size, 0.2 × 150 mm) with isocratic elution at 0.2 ml/min with 3.75% MeCN and 0.1% TFA in water (solvent A) for 15 min. After each run the column was washed by elution with 50% tetrahydrofuran in 0.1% TFA in water for 20 min and reequilibrated by elution with solvent A at 0.4 ml/min for 15 min. Pentosidine was detected by in-line fluorimetry; excitation wavelength 320 nm, emission wavelength 365 nm [35].