High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach.
Stable isotope dilution-selective reaction monitoring-mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Junying
J
Wei
Chen
C
Ding
Jiao
J
Zhang
Wei
W
Mi
Yan
Y
Zhao
Mingwei
M
Liu
Tianyi
T
Fu
Yangjun
Y
Zhang
Wantao
W
Ying
Yun
Y
Cai
Jun
J
Qin
Xiaohong
X
Qian
D000595:Amino Acid Sequence; D000818:Animals; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation / Q000379:methods*; D005982:Glutathione Transferase / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods
D010455:Peptides; D011506:Proteins; D005982:Glutathione Transferase