Table 1.

Selection of fixed and variable modifications in peptide characterization and quantitation.

Experimental Procedures	(1a) Protein Identification and Quantification in TMT Experiments	(1b) Protein Identification and Quantification in TMT Experiments	(2a) Estimation of TMT Ratio Compression	(2b) Estimation of TMT Ratio Compression	(3) Endocytosis Assay Using SILAC
Purposes or studies	Relative quantitation of 6-plex TMT peptides in studies of i) +/−WP, D9 -> D15 ii) +/−WP, D9 ->D22 iii) –WP, D9 -> D21 iv) WP endocytosis using TMT v) Caco-2 proteins with UPS-2 spiked	Labeling efficiency of TMT 6-plex (the number of peptides without TMT versus the number of peptides with TMT from (1a)	Relative quantitation of regular 6-plex TMT peptides under the presence of Lys-8, Arg-10-containing 6-plex TMT peptides	Relative quantitation of Lys-8, Arg-10-containing 6-plex TMT peptides under the presence of regular 6-plex TMT peptides	Endocytosis of WP by a Caco-2 monolayer
Types of peptide sequences for analysis	Regular tryptic peptides with TMT labels	Regular tryptic peptides without TMT labels	Regular tryptic peptides with TMT labels	Lys8, Arg10-containing tryptic peptides with TMT labels	Regular (L) versus Lys8, Arg10-containing (H) tryptic peptides
Fixed modifications	Carbamidomethyl (C), TMT6plex (N-term), TMT6plex (K)	Carbamidomethyl (C)	Carbamidomethyl (C), TMT6plex (N-term), TMT6plex (K)	Carbamidomethyl (C), Label:13C(6)15N(4) (R), TMT6plex (N-term), Label:13C(6)15N(2)+TMT6plex (K)	Carbamidomethyl (C)
Variable modifications	Acetyl (N-term), Deamidated (NQ), Oxidation (M)	Acetyl (N-term), Deamidated (NQ), Oxidation (M)	Acetyl (N-term), Deamidated (NQ), Oxidation (M)	Acetyl (N-term), Deamidated (NQ), Oxidation (M)	Acetyl (N-term), Deamidated (NQ), Oxidation (M), SILAC K+8 R+10
Quantitation method in Mascot	TMT 6plex	N/A	TMT 6plex	TMT 6plex	L/H protein ratios from extracted ion chromatograms