Nuts and Bolts of Protein Quantification by Online Trypsin Digestion Coupled LC-MS/MS Analysis

ABSTRACTThe data-independent acquisition (DIA) performed in the latest high-resolution, high-speed mass spectrometers offers a powerful analytical tool for biological investigations. The DIA mass spectrometry (MS) combined with the isotopic labeling approach holds a particular promise for increasing the multiplexity of DIA-MS analysis, which could assist the relative protein quantification and the proteome-wide turnover profiling. However, the wide isolation windows employed in conventional DIA methods lead to a limited efficiency in identifying and quantifying isotope-labelled peptide pairs. Here, we optimized a high-selectivity DIA-MS named BoxCarmax that supports the analysis of complex samples, such as those generated from Stable isotope labeling by amino acids in cell culture (SILAC) and pulse SILAC (pSILAC) experiments. BoxCarmax enables multiplexed acquisition at both MS1- and MS2-levels, through the integration of BoxCar and MSX features, as well as a gas-phase separation strategy. We found BoxCarmax modestly increased the identification rate for label-free and labeled samples but significantly improved the quantitative accuracy in SILAC and pSILAC samples. We further applied BoxCarmax in studying the protein degradation regulation during serum starvation stress in cultured cells, revealing valuable biological insights. Our study offered an alternative and accurate approach for the MS analysis of protein turnover and complex samples.

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Characterization of succinimide stability during trypsin digestion for LC-MS analysis

Analytical Biochemistry ◽

10.1016/j.ab.2017.03.005 ◽

2017 ◽

Vol 526 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Christine Nowak ◽

Gomathinayagam Ponniah ◽

Alyssa Neill ◽

Hongcheng Liu

Keyword(s):

Trypsin Digestion ◽

Ms Analysis

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Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster

10.7287/peerj.preprints.1677v1 ◽

2016 ◽

Author(s):

Perumal Subramanian ◽

Jaime J Jayapalan ◽

Puteri Abdul-Rahman ◽

Manjula Arumugam ◽

Onn Hashim

Keyword(s):

Protein Synthesis ◽

Drosophila Melanogaster ◽

Protein Profile ◽

Redox Homeostasis ◽

Biological Clock ◽

Fruit Fly ◽

Trypsin Digestion ◽

Diurnal Rhythms ◽

Protein Accumulation ◽

Ms Analysis

Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie rhythmic physiology in the fruit fly, Drosophila melanogaster. Self-sustained autonomous circadian oscillations were documented all over the organs of the fly. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. We have used the whole fly for the proteomic study as performed in typical proteotypic peptide (PTP) studies and followed the same protocol with trypsin digestion. Total protein collected from fly samples harvested at 4h intervals over the 24-h period were subjected to two dimensional (2-D) gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. A significant variation in the expression of 88 proteins over the 24-h period was observed. Our present results suggested that the synthesis/regulation of numerous proteins is regulated by the biological clock in D. melanogaster. Relatively higher number of proteins was upregulated during nighttime as compared to daytime. Conclusion. As these rhythmically varying proteins/enzymes involve in metabolism, muscle activities, ion channels, protein synthesis, redox homeostasis and apoptosis our results indicate that these cellular processes could be regulated at the level of temporal expression of protein profile.

Download Full-text

Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster

10.7287/peerj.preprints.1677 ◽

2016 ◽

Author(s):

Perumal Subramanian ◽

Jaime J Jayapalan ◽

Puteri Abdul-Rahman ◽

Manjula Arumugam ◽

Onn Hashim

Keyword(s):

Protein Synthesis ◽

Drosophila Melanogaster ◽

Protein Profile ◽

Redox Homeostasis ◽

Biological Clock ◽

Fruit Fly ◽

Trypsin Digestion ◽

Diurnal Rhythms ◽

Protein Accumulation ◽

Ms Analysis

Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie rhythmic physiology in the fruit fly, Drosophila melanogaster. Self-sustained autonomous circadian oscillations were documented all over the organs of the fly. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. We have used the whole fly for the proteomic study as performed in typical proteotypic peptide (PTP) studies and followed the same protocol with trypsin digestion. Total protein collected from fly samples harvested at 4h intervals over the 24-h period were subjected to two dimensional (2-D) gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. A significant variation in the expression of 88 proteins over the 24-h period was observed. Our present results suggested that the synthesis/regulation of numerous proteins is regulated by the biological clock in D. melanogaster. Relatively higher number of proteins was upregulated during nighttime as compared to daytime. Conclusion. As these rhythmically varying proteins/enzymes involve in metabolism, muscle activities, ion channels, protein synthesis, redox homeostasis and apoptosis our results indicate that these cellular processes could be regulated at the level of temporal expression of protein profile.

Download Full-text